Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 76-86).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Abstract Approximately 5–10% of all breast cancer cases are estimated to result from a hereditary predisposition to the disease. Currently no more than 25–30% of these familial cases can be explained by mutations in the known susceptibility genes, BRCA1 and BRCA2 being the major ones. Additional predisposing genes are therefore likely to be discovered. This study evaluates whether germline alterations in three BRCA1-associated genes, BACH1 (i.e. BRIP1/FANCJ), BARD1 and TOPBP1, contribute to familial breast cancer. Altogether 214 Finnish patients having breast and/or ovarian cancer were analysed for germline mutations in the BACH1 gene. Nine alterations were observed, four of which located in the protein-encoding region. The previously unidentified Pro1034Leu was considered a possible cancer-associated alteration as it appeared with two-fold higher frequency among cancer cases compared to controls. All the other observed alterations were classified as harmless polymorphisms. Mutation analysis of the BARD1 gene among 126 Finnish patients having family history of breast and/or ovarian cancer revealed seven alterations in the protein-encoding region. The Cys557Ser alteration was seen at an elevated frequency among familial cancer cases compared to controls (p = 0.005, odds ratio [OR] 4.2, 95% confidence interval [CI] 1.7–10.7). The other alterations appeared to be harmless polymorphisms. To evaluate further the possible effect of Cys557Ser on cancer risk, a large case-control study was performed, consisting of 3,956 cancer patients from the Nordic countries. The highest prevalence of Cys557Ser was found among breast and ovarian cancer patients from BRCA1/BRCA2 mutation-negative families (p < 0.001, OR 2.6, 95% CI 1.7–4.0). In contrast, no significant association with male breast cancer, ovarian, colorectal or prostate cancer was observed. The current study is the first evaluating the role of TOPBP1 mutations in familial cancer predisposition. The analysis of 125 Finnish patients having breast and/or ovarian cancer revealed one putative pathogenic alteration. The commonly occurring Arg309Cys allele was observed at a significantly higher frequency among familial cancer cases compared to controls (p = 0.002, OR 2.4, 95% CI 1.3–4.2). The other 18 alterations observed were classified as harmless polymorphisms.

Abstract In Western countries, breast and ovarian cancer are among the most frequent malignancies affecting women. Approximately 5–10% of the cases in the general population have been suggested to be attributed to inherited disease susceptibility. BRCA1 and BRCA2 are the main genes associated with predisposition to breast and ovarian cancer. Mutations in these two genes explain a major part of the families displaying a large number of early-onset breast and/or ovarian cancers, but at least one third of the cases appear to be influenced by other, as yet unidentified genes. Therefore, it is likely that defects in other cancer predisposing genes, perhaps associated with lower disease penetrance and action in a polygenic context, will also be discovered. In the present study, the contribution of germline mutations in putative breast and/or ovarian cancer susceptibility genes, based on their biological function, has been investigated in Finnish breast cancer families. The role of large genomic deletions or other rearrangements in the BRCA1 and BRCA2 genes was evaluated by Southern blot analysis, and mutation analysis of TP53, RAD51, the BRC repeats of BRCA2, and 53BP1 was performed by conformation sensitive gel electrophoresis and DNA sequencing. Germline TP53 mutations were searched for in 108 Finnish breast cancer families without BRCA1 or BRCA2 alterations. In this study, the pathogenic TP53 germline mutation, Arg248Gln, was identified in only one family. This family showed a strong family history of breast cancer and other cancers also fulfilling the criteria for Li-Fraumeni-like syndrome. Germline TP53 mutations are expected to be found in cancer families with clinical features seen in Li-Fraumeni or Li-Fraumeni-like syndromes. In this study, large deletions in BRCA1 and BRCA2 were not observed in 82 breast and/or ovarian cancer families. Likewise, no disease-related aberrations were detected in RAD51, the BRC repeats of BRCA2 or 53BP1 in the 126 breast and/or ovarian cancer families studied. The obtained results were validated by comparing to the occurrence in 288–300 female cancer-free control individuals. These results do not support the hypothesis that alterations in these particular genomic regions play a significant role in breast cancer predisposition in Finland. Thus, there are still genes to be discovered to explain the molecular background of breast cancer.

Orientadores: Fabíola Traina, Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T21:37:05Z (GMT). No. of bitstreams: 1 Campos_PauladeMelo_D.pdf: 6042513 bytes, checksum: b12134db0721f177eb0c2116e7fdf5e2 (MD5) Previous issue date: 2015 / Resumo: As neoplasias mieloproliferativas crônicas BCR-ABL1 negativas (NMP) apresentam como característica comum a ocorrência de proliferação celular exacerbada, mantendo a capacidade de diferenciação mieloide terminal. Em parcela significativa dos casos, a ativação da proliferação celular ocorre pelo aumento da atividade tirosinoquinase de proteínas específicas. Entretanto, a heterogeneidade molecular observada nos pacientes e as respostas clínicas insatisfatórias observadas em parte dos casos com os tratamentos vigentes sugerem que mecanismos adicionais, como interações proteicas não descritas e novas mutações, possam estar envolvidos na fisiopatologia destas neoplasias. Neste trabalho, objetivamos estudar as vias de ativação tirosinoquinase em policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) (subprojeto 1), e em mastocitose sistêmica (subprojeto 2). O foco principal do subprojeto 1 consistiu em avaliar, em NMP, a associação JAK2/IRS2, previamente descrita em células não hematológicas após estímulo, bem como o envolvimento de IRS2 em vias de proliferação celular e apoptose. Utilizamos modelos de linhagens celulares leucêmicas humanas com JAK2 mutado (JAK2V617F) e JAK2 selvagem (JAK2WT), submetendo-as a inibição gênica por shRNA entregue por lentivírus e ao tratamento com o inibidor seletivo de JAK1/2 ruxolitinib. As células foram então submetidas à avaliação da viabilidade celular por MTT, e da apoptose por citometria de fluxo (anexina V/PI e caspase-3) e por imunobloting (caspase-3 clivada). A expressão do mRNA de IRS2 foi avaliada por PCR em tempo real em amostras de células CD34+ de sangue periférico de 99 pacientes com diagnóstico de PV, TE e MFP, e em 28 doadores normais. Através de imunoprecipitação e microscopia confocal, observamos a associação constitutiva entre JAK2 e IRS2 nas células JAK2V617F, mas não nas células JAK2WT. Em células JAK2V617F, a inibição de IRS2 por lentivírus diminuiu significativamente a fosforilação de STAT5, reduziu a viabilidade celular, aumentou as taxas de apoptose, e potencializou os efeitos de ruxolitinib. Não houve mudanças nas taxas de viabilidade celular e apoptose nas células JAK2WT inibidas para IRS2. A expressão do mRNA de IRS2 foi significativamente maior nos pacientes com TE em relação aos doadores normais, e em pacientes com NMP portadores da mutação JAK2V617F em relação aos pacientes com JAK2WT. Estas evidências sugerem que IRS2 possa participar das vias de sinalização celular nas NMP através de interação direta com JAK2. A inibição farmacológica de IRS2, isoladamente ou em conjunto com ruxolitinib, é ferramenta potencial no tratamento de pacientes com PV, TE e MFP. No subprojeto 2, nosso objetivo consistiu em investigar mutações no gene KIT em um caso de mastocitose sistêmica familiar seguido em nosso serviço, em que mãe (caso 1) e filha (caso 2) apresentavam extensa infiltração cutânea e da medula óssea por mastócitos, bem como avaliar a sensibilidade dos mastócitos neoplásicos ao tratamento com os inibidores de tirosinoquinase (ITK) imatinibe, dasatinibe e PKC412. Através de sequenciamento por Sanger, identificamos a mutação KITK509I em células de medula óssea total, CD3+ de sangue periférico e mucosa oral de ambas pacientes. Os pais do caso 1 apresentaram KIT selvagem. O tratamento in vitro por 4, 8 ou 12 dias de células totais de medula óssea dos casos 1 e 2 com os ITK avaliados resultou em menor viabilidade celular, avaliada por MTT, e em redução da fosforilação de P70S6K com todas as drogas testadas. Entretanto, apenas o imatinibe evidenciou resposta consistente na indução de apoptose. Foi iniciado tratamento dos casos 1 e 2 com imatinibe 400mg/dia via oral. Três meses após o início, houve normalização da pele e da medula óssea; após dois anos de seguimento, as pacientes mantêm-se em remissão. Embora rara, a mutação KITK509I deve ser pesquisada em todos os casos de mastocitose sistêmica familiar. O imatinibe pode ser considerado como droga de primeira escolha nestes casos / Abstract: The BCR-ABL1 negative chronic myeloproliferative neoplasms (MPN) are characterized by increased cellular proliferation with preserved terminal myeloid differentiation. In most cases, the activation of cell proliferation is caused by an increased tyrosine kinase activity of specific proteins. However, patients' molecular heterogeneity and the incomplete clinical responses observed in part of the cases using the current treatments suggest that additional mechanisms, such as unknown protein interactions and new mutations, can be involved in the pathophysiology of MPN. In this study, our main goal was to investigate tyrosine kinase activation pathways in polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) (subproject 1), and in systemic mastocytosis (subproject 2). The focus of subproject 1 consisted in the investigation of JAK2/IRS2 association in MPN, already described in non-hematological cells following extrinsic stimulus, and in evaluating IRS2 function in MPN cell proliferation and apoptosis. JAK2 wild-type (JAK2WT) and JAK mutated (JAK2V617F) human leukemia cell lines were transduced with lentivirus-mediated shRNA targeting IRS2, and treated with vehicle (DMSO) or with the selective JAK1/2 inhibitor ruxolitinib. Cells were then submitted to evaluation of cell viability (MTT) and apoptosis (anexin V/PI and caspase-3 by flow cytometry, and cleaved caspase-3 by immunoblotting). IRS2 mRNA expression was evaluated by real time quantitative PCR in CD34+ peripheral blood cells of 99 patients with PV, ET and PMF, and in 28 healthy donors. Through immunoprecipitation/immunobloting and confocal microscopy, we observed the constitutive JAK2/IRS2 association in JAK2V617F cells, but not in JAK2WT cell lines. In JAK2V617F, IRS2 silencing significantly decreased phospho-STAT5, reduced cell viability, induced apoptosis, and potentiated the effects of ruxolitinib treatment. No differences in cell viability and apoptosis ratios were observed in IRS2 silenced JAK2WT cells. IRS2 mRNA expression was significantly higher in ET patients when compared to healthy donors, and in patients harboring JAK2V617F mutation in relation to JAK2WT. These evidence suggest that IRS2 participate in MPN cell signaling pathways through its interaction with JAK2. IRS2 pharmacological inhibition, alone or in combination with ruxolitinib, may be a potential tool in the treatment of PV, ET and PMF patients. In subproject 2, our main goal was to seek for KIT mutations in a case of systemic familial mastocytosis, followed in our outpatient clinics, in which the mother (case 1) and the daughter (case 2) had extensive skin and bone marrow infiltration by mast cells. Also, we aimed to evaluate in vitro sensitivity of neoplastic mast cells to the treatment with the tyrosine kinase inhibitors (TKI) imatinibe, dasatinibe and PKC412. Using Sanger sequencing analysis, we identified the KITK509I mutation in total bone marrow, peripheral blood CD3+ and oral mucosa cells in both patients. The parents of case 1 had wild type KIT. The in vitro treatment of total bone marrow cells of cases 1 and 2 with TKI for 4, 8 and 12 days resulted in reduced cell viability, as evaluated by MTT, and in reduced phosphorylation of P70S6K for all tested drugs. However, only imatinibe consistently induced apoptosis in both cases. Patients were started on imatinibe 400mg orally per day. Three months following imatinibe treatment, there was a complete reversion of skin and bone marrow mast cells infiltration; after two years of follow-up, cases 1 and 2 remain in complete remission of the systemic mastocytosis. Although rare, KITK509I mutation should be investigated in all cases of familial systemic mastocytosis. Imatinibe is a good first choice for the treatment of these cases / Doutorado / Fisiopatologia Médica / Doutora em Ciências

En France, le cancer de sein héréditaire représente environ 2500 nouveaux cas par an, dont prés de la moitié est attribuée à la mutation du gène BRCA1.La recherche de la mutation par biologie moléculaire est un travail fastidieux, coûteux et long (8 mois d’attente environ actuellement).Pour trouver une solution à ce délai, nous avons étudié en immunohistochimie une série initiale de 21 anticorps répartis en 5 groupes : anticorps antiBrca1 du commerce, liés à la perte de l’inactivation de l’X, liés à la signature basale ou myoépithéliale, anticorps dits classiques du cancer de sein et finalement dérivés de signatures établies par cDNAarray.Nous avons utilisé la technique de’ tissue microarrays’ en utilisant de manière comparative une population de 27 cas de cancer de sein présentant une mutation germinale de BRCA1, et 81 cas témoins de cancer de sein sporadiques appariés à l’âge, ainsi qu’à des lignées cellulaires d’origine mammaires. Dans une deuxième série indépendante de validation nous avons appliqué les résultats obtenus de la première série sur 28 cas de cancer mammaire muté, et 28 cas du cancer mammaire sporadique dans les mêmes conditions initiales.Nos résultats montrent pour la première fois sur des tissus tumoraux une probabilité forte d’une association entre la mutation Brca1 et la perte de l’inactivation de l’X ; confirment la valeur de MS110 comme un bon anticorps prédictif d’une mutation de Brca1 ; apportent un argument pour une participation myoépithéliale dans l’oncogenèse de cancer mammaire Brca1 muté; appuient la relation entre ce dernier et les récepteurs RE,RP ainsi que P53 , Bcl2,Ki67 et valident en protéomique la valeur discriminant de CDC47 correspondant à un des gènes de la signature génomique.Après confirmation des mêmes résultats dans la série de validation, nous soutenons en analyses multivariés un modèle qui comprend seulement Grade 3, MS110, Lys27H3 négative, Vimentine et KI67 positive. Cette équation correspond à une sensibilité de 82% et spécificité de 81% et propose une approche rapide économique de pré- ciblage de la mutation Brca1 ; ce qui améliorait la prise en charge préventive, thérapeutique et globale des patients et leurs familles. / Family structure, lack of reliable information, cost and delay are usual concerns faced with when deciding to perform BRCA analyses. Testing the breast cancer tissues with four antibodies (MS110, lys27H3, Vimentin, KI67) in addition to grade evaluation enabled to rapidly select patients to carry out genetic testing identification. We constituted an initial breast cancer tissue micro-array, considered as a learning set comprising 27 BRCA1 and 81 sporadic tumours. A second independent validation set of 28 BRCA1 tumours was matched to 28 sporadic tumours using the same original conditions.We have investigated morphological parameters and 21 markers by immunohistochemistry.A logistic regression model was used to select the minimal number of markers providing the best model to predict BRCA1 status. The model was applied to the validation set to estimate specificity and sensibility.In the initial set, the univariate analysis identified 11 markers significantly associated with BRCA1 status. Then the best multivariate model comprised only Grade 3, MS110, Lys27H3, Vimentin and KI67. When applied to the validation set, BRCA1 tumours were correctly classified with a sensitivity of 83% and a specificity of 81%. The performance of this model was superior when compared to other profiles.This work offers a new rapid and economic method for the pre-screening of patients at high risk of being BRCA1mutation carriers, then to guide genetic testing, and finally to provide appropriate preventive measure, advices and treatments including targeted therapy to patients and their families.

As maiores taxas de incidência de câncer de mama ocorrem em mulheres idosas, que apresentam tumores com expressão de receptores de estrógeno e/ou progesterona, de baixo estadiamento e menor taxa de proliferação, se comparado com as jovens. Um dos fatores de predisposição ao câncer de mama é mutação germinativa nos genes BRCA1 ou BRCA2, que podem compreender entre 5-10% das pacientes diagnosticadas. A grande maioria dos casos são ditos esporádicos, em que não há como estabelecer um único fator determinante. Dentre o escopo de possíveis causas estão as mutações somáticas, acumuladas no tecido mamário ao longo da vida. A identificação destas mutações permite melhor compreensão da carcinogênese e possibilita a criação de tratamentos cada vez mais personalizados. O gene PIK3CA, por exemplo, já está determinado como driver (responsáveis pela obtenção de vantagem seletiva de um determinado clone) para câncer de mama. As mutações patogênicas que ocorrem neste gene levam a ativação da via de Akt/mTOR, entre outras, que mantém o ciclo celular ativo. Um gene que vem sendo estudado recentemente é o PRKD1, cujas funções parecem estar ligadas à manutenção do fenótipo epitelial das células do tecido mamário. Assim, o objetivo desse trabalho identificar mutações germinativas nos genes BRCA1 e BRCA2, analisando também o histórico familiar para câncer de mama/ovário/próstata, e mutações somáticas no gene PRKD1 em pacientes pós menopausadas,. Foram incluídas quarenta e nove pacientes diagnosticadas com carcinoma ductal invasivo em idade superior a 54 anos, que preenchessem critérios da NCCN (National Comprehensive Cancer Network) para Síndrome de Câncer de Mama e/ou Ovário Hereditário e tinham disponível um fragmento tumoral emblocado em parafina coletado na ausência de tratamento neo adjuvante. A extração de DNA foi realizada a partir do sangue periférico para sequenciamento de BRCA1 e BRCA2, realizado através da plataforma Ion Torrent(TM) ou pelo método de Sanger. Os resultados obtidos por Ion Torrent(TM) foram analisados, primeiramente, através da ferramenta online Ion Reporter e os de Sanger através do programa Mutation Surveyor v.3.20. Para a caractetização das variantes encontradas foram utilizados: os bancos de dados BIC, LOVD, LOVD-IARC, UMD e ClinVar além dos preditores in silico da conservação dos aminoácidos entre as espécies Polyphen-2, SIF, Provean e AlignGVGD e do preditor de efeito no splicing HSF e bancos de dados de frequência alélica ExAC, 1000 genomas e NHLBI GO Exome Sequencing Project, seguindo os critérios da American College of Medical Genetics and Genomics em conjunto com a Association for Molecular Pathology. Para caracterização de mutação somática do gene PRKD1 determinou-se duas regiões de maior importância para serem sequenciadas: Ser738/Ser742 e Ser910 que fosforilam o domínio quinase da proteína, ativando-o. Vinte e três amostras tumorais tiveram DNA extraído. Também foi realizada uma análise das informações sobre PRKD1 do banco de dados COSMIC (Catalogue of Somatic Mutations in Cancer) e a construção de curvas de sobrevida (Kaplan-Meier) da expressão de PRKD1 utilizando a ferramenta online KM Plotter. A idade mediana das pacientes foi de 62 anos ao diagnóstico e de 64 anos na época de inclusão no estudo. A maioria tinha tumores de grau histológico II (63,27%), estádio clinico II (20%) e do subtipo luminal B (53,06%). Trinta e duas relataram parentes de primeiro grau afetados com câncer de mama/ovário/ próstata. Trinta e oito pacientes tiveram sequenciamento completo de BRCA1 e BRCA2 por Ion Torrent(TM) e onze tiveram sequenciamento parcial de BRCA1 e BRCA2 por Sanger. Variantes patogênicas foram encontradas em quatro pacientes (BRCA1=2/BRCA2=2). Uma nova variante missense foi identificada em BRCA2: c.3371A > G (p.Q1124R). Para o sequenciamento de PRKD1 quinze foram sequenciadas para Ser910 e de oito foi possível analisar o resultado. Nenhuma variante patogênica foi encontrada. Os dados obtidos sobre PRKD1 no COSMIC foram: de 2773 amostras, em apenas 15 (0,54%) foram identificadas mutações em PRKD1, 46% (7/15) provém de mulheres com idade superior a 55 anos e subtipo molecular Luminal. PRKD1 apresenta maiores frequência de mutação em câncer de intestino grosso (4,22%) e pele (4,02%). As curvas de sobrevida construídas no KM Plotter demonstram a alta expressão do gene parece ter impacto positivo na sobrevida das pacientes. Apesar da baixa frequência de mutações no PRKD1 este gene, outros dados demonstram que parece ter um papel de gene supressor de tumor no câncer de mama, que deve ser inibido de através de outros mecanismos como metilaçao de DNA / The highest rates of breast cancer incidence occur in elderly women, who present estrogen and / or progesterone receptor tumors, with a low clinical staging and lower proliferation rate compared to the young women. One of the factors predisposing to breast cancer is germline mutation in the BRCA1 or BRCA2 genes, which may comprise between 5-10% of the diagnosed patients. The vast majority of cases are said to be sporadic, in which there is no way to establish a single determining factor. Among the scope of possible causes are somatic mutations, accumulated in the breast tissue throughout life. The identification of these mutations allows a better understanding of carcinogenesis and enables the creation of increasingly personalized treatments. The PIK3CA gene, for example, is already determined as a driver (responsible for the selective advantage of a particular clone) for breast cancer. The pathogenic mutations that occur in this gene lead to the activation of Akt / mTOR pathway, among others, which keeps the cell cycle active. One gene that has recently been studied is PRKD1, whose functions seem to be linked to the maintenance of the epithelial phenotype of the mammary tissue cells. Thus, the objective of this work was to identify germline mutations in BRCA1 and BRCA2 genes, also analyzing the family history for breast / ovarian / prostate cancer, and somatic mutations in the PRKD1 gene in postmenopausal patients. Forty-nine patients diagnosed with ipsilateral ductal carcinomas over the age of 54 years who completed NCCN (National Comprehensive Cancer Network) criteria for Breast Cancer and / or Hereditary Ovarian Syndrome and had a tumor paraffin embedded in paraffin collected in the absence of neo adjuvant treatment available. DNA extraction was performed from the peripheral blood for sequencing of BRCA1 and BRCA2, performed through the Ion Torrent (TM) platform or by the Sanger method. The results obtained by Ion Torrent (TM) were first analyzed through the online tool Ion Reporter and those by Sanger through the program Mutation Surveyor v.3.20. The BIC, LOVD, LOVD-IARC, UMD and ClinVar databases were used in addition to the in silico predictors of amino acid conservation among Polyphen-2, SIF, Provean and AlignGVGD species and the effect predictor in the HSF splicing and allelic frequency databases ExAC, 1000 genomes and the NHLBI GO Exome Sequencing Project, following the criteria of the American College of Medical Genetics and Genomics in conjunction with the Association for Molecular Pathology. In order to characterize the somatic mutation of the PRKD1 gene, we determined two regions of greater importance to be sequenced: Ser738 / Ser742 and Ser910 that phosphorylate the protein kinase domain, activating it. Twenty-three tumor samples had DNA extracted. An analysis of PRKD1 information from the COSMIC (Catalog of Somatic Mutations in Cancer) database and the construction of survival curves (Kaplan-Meier) for PRKD1 expression using the online KM Plotter tool was also performed. The median age of the patients was 62 years at diagnosis and 64 years at the time of inclusion in the study. Most of them had tumors of histological grade II (63.27%), clinical stage II (20%) and molecular subtype luminal B (53.06%). Thirty-two reported first-degree relatives affected with breast / ovarian / prostate cancer. Thirty-eight patients had BRCA1 and BRCA2 complete sequencing by Ion Torrent (TM) and eleven had BRCA1 and BRCA2 partial sequencing by Sanger. Pathogenic variants were found in four patients (BRCA1 = 2 / BRCA2 = 2). For PRKD1 sequencing, fifteen patients tumors were sequenced for Ser910 and in eight samples it was possible to analyze the result. No pathogenic variant was found. The data obtained on PRKD1 in COSMIC were: from 2773 samples, in only 15 (0.54%) mutations were identified in PRKD1, 46% (7/15) came from women aged over 55 years and had tumor molecular subtype Luminal. PRKD1 shows higher mutation frequency in cancer of the large intestine (4.22%) and skin (4.02%). The survival curves constructed in KM Plotter demonstrate the high expression of the gene seems to have a positive impact on the patients survival . Despite the low frequency of mutations in PRKD1 gene, other data demonstrate that it appears to play a role of tumor suppressor gene in breast cancer, which must be inhibited by other mechanisms such as DNA methylation

As maiores taxas de incidência de câncer de mama ocorrem em mulheres idosas, que apresentam tumores com expressão de receptores de estrógeno e/ou progesterona, de baixo estadiamento e menor taxa de proliferação, se comparado com as jovens. Um dos fatores de predisposição ao câncer de mama é mutação germinativa nos genes BRCA1 ou BRCA2, que podem compreender entre 5-10% das pacientes diagnosticadas. A grande maioria dos casos são ditos esporádicos, em que não há como estabelecer um único fator determinante. Dentre o escopo de possíveis causas estão as mutações somáticas, acumuladas no tecido mamário ao longo da vida. A identificação destas mutações permite melhor compreensão da carcinogênese e possibilita a criação de tratamentos cada vez mais personalizados. O gene PIK3CA, por exemplo, já está determinado como driver (responsáveis pela obtenção de vantagem seletiva de um determinado clone) para câncer de mama. As mutações patogênicas que ocorrem neste gene levam a ativação da via de Akt/mTOR, entre outras, que mantém o ciclo celular ativo. Um gene que vem sendo estudado recentemente é o PRKD1, cujas funções parecem estar ligadas à manutenção do fenótipo epitelial das células do tecido mamário. Assim, o objetivo desse trabalho identificar mutações germinativas nos genes BRCA1 e BRCA2, analisando também o histórico familiar para câncer de mama/ovário/próstata, e mutações somáticas no gene PRKD1 em pacientes pós menopausadas,. Foram incluídas quarenta e nove pacientes diagnosticadas com carcinoma ductal invasivo em idade superior a 54 anos, que preenchessem critérios da NCCN (National Comprehensive Cancer Network) para Síndrome de Câncer de Mama e/ou Ovário Hereditário e tinham disponível um fragmento tumoral emblocado em parafina coletado na ausência de tratamento neo adjuvante. A extração de DNA foi realizada a partir do sangue periférico para sequenciamento de BRCA1 e BRCA2, realizado através da plataforma Ion Torrent(TM) ou pelo método de Sanger. Os resultados obtidos por Ion Torrent(TM) foram analisados, primeiramente, através da ferramenta online Ion Reporter e os de Sanger através do programa Mutation Surveyor v.3.20. Para a caractetização das variantes encontradas foram utilizados: os bancos de dados BIC, LOVD, LOVD-IARC, UMD e ClinVar além dos preditores in silico da conservação dos aminoácidos entre as espécies Polyphen-2, SIF, Provean e AlignGVGD e do preditor de efeito no splicing HSF e bancos de dados de frequência alélica ExAC, 1000 genomas e NHLBI GO Exome Sequencing Project, seguindo os critérios da American College of Medical Genetics and Genomics em conjunto com a Association for Molecular Pathology. Para caracterização de mutação somática do gene PRKD1 determinou-se duas regiões de maior importância para serem sequenciadas: Ser738/Ser742 e Ser910 que fosforilam o domínio quinase da proteína, ativando-o. Vinte e três amostras tumorais tiveram DNA extraído. Também foi realizada uma análise das informações sobre PRKD1 do banco de dados COSMIC (Catalogue of Somatic Mutations in Cancer) e a construção de curvas de sobrevida (Kaplan-Meier) da expressão de PRKD1 utilizando a ferramenta online KM Plotter. A idade mediana das pacientes foi de 62 anos ao diagnóstico e de 64 anos na época de inclusão no estudo. A maioria tinha tumores de grau histológico II (63,27%), estádio clinico II (20%) e do subtipo luminal B (53,06%). Trinta e duas relataram parentes de primeiro grau afetados com câncer de mama/ovário/ próstata. Trinta e oito pacientes tiveram sequenciamento completo de BRCA1 e BRCA2 por Ion Torrent(TM) e onze tiveram sequenciamento parcial de BRCA1 e BRCA2 por Sanger. Variantes patogênicas foram encontradas em quatro pacientes (BRCA1=2/BRCA2=2). Uma nova variante missense foi identificada em BRCA2: c.3371A > G (p.Q1124R). Para o sequenciamento de PRKD1 quinze foram sequenciadas para Ser910 e de oito foi possível analisar o resultado. Nenhuma variante patogênica foi encontrada. Os dados obtidos sobre PRKD1 no COSMIC foram: de 2773 amostras, em apenas 15 (0,54%) foram identificadas mutações em PRKD1, 46% (7/15) provém de mulheres com idade superior a 55 anos e subtipo molecular Luminal. PRKD1 apresenta maiores frequência de mutação em câncer de intestino grosso (4,22%) e pele (4,02%). As curvas de sobrevida construídas no KM Plotter demonstram a alta expressão do gene parece ter impacto positivo na sobrevida das pacientes. Apesar da baixa frequência de mutações no PRKD1 este gene, outros dados demonstram que parece ter um papel de gene supressor de tumor no câncer de mama, que deve ser inibido de através de outros mecanismos como metilaçao de DNA / The highest rates of breast cancer incidence occur in elderly women, who present estrogen and / or progesterone receptor tumors, with a low clinical staging and lower proliferation rate compared to the young women. One of the factors predisposing to breast cancer is germline mutation in the BRCA1 or BRCA2 genes, which may comprise between 5-10% of the diagnosed patients. The vast majority of cases are said to be sporadic, in which there is no way to establish a single determining factor. Among the scope of possible causes are somatic mutations, accumulated in the breast tissue throughout life. The identification of these mutations allows a better understanding of carcinogenesis and enables the creation of increasingly personalized treatments. The PIK3CA gene, for example, is already determined as a driver (responsible for the selective advantage of a particular clone) for breast cancer. The pathogenic mutations that occur in this gene lead to the activation of Akt / mTOR pathway, among others, which keeps the cell cycle active. One gene that has recently been studied is PRKD1, whose functions seem to be linked to the maintenance of the epithelial phenotype of the mammary tissue cells. Thus, the objective of this work was to identify germline mutations in BRCA1 and BRCA2 genes, also analyzing the family history for breast / ovarian / prostate cancer, and somatic mutations in the PRKD1 gene in postmenopausal patients. Forty-nine patients diagnosed with ipsilateral ductal carcinomas over the age of 54 years who completed NCCN (National Comprehensive Cancer Network) criteria for Breast Cancer and / or Hereditary Ovarian Syndrome and had a tumor paraffin embedded in paraffin collected in the absence of neo adjuvant treatment available. DNA extraction was performed from the peripheral blood for sequencing of BRCA1 and BRCA2, performed through the Ion Torrent (TM) platform or by the Sanger method. The results obtained by Ion Torrent (TM) were first analyzed through the online tool Ion Reporter and those by Sanger through the program Mutation Surveyor v.3.20. The BIC, LOVD, LOVD-IARC, UMD and ClinVar databases were used in addition to the in silico predictors of amino acid conservation among Polyphen-2, SIF, Provean and AlignGVGD species and the effect predictor in the HSF splicing and allelic frequency databases ExAC, 1000 genomes and the NHLBI GO Exome Sequencing Project, following the criteria of the American College of Medical Genetics and Genomics in conjunction with the Association for Molecular Pathology. In order to characterize the somatic mutation of the PRKD1 gene, we determined two regions of greater importance to be sequenced: Ser738 / Ser742 and Ser910 that phosphorylate the protein kinase domain, activating it. Twenty-three tumor samples had DNA extracted. An analysis of PRKD1 information from the COSMIC (Catalog of Somatic Mutations in Cancer) database and the construction of survival curves (Kaplan-Meier) for PRKD1 expression using the online KM Plotter tool was also performed. The median age of the patients was 62 years at diagnosis and 64 years at the time of inclusion in the study. Most of them had tumors of histological grade II (63.27%), clinical stage II (20%) and molecular subtype luminal B (53.06%). Thirty-two reported first-degree relatives affected with breast / ovarian / prostate cancer. Thirty-eight patients had BRCA1 and BRCA2 complete sequencing by Ion Torrent (TM) and eleven had BRCA1 and BRCA2 partial sequencing by Sanger. Pathogenic variants were found in four patients (BRCA1 = 2 / BRCA2 = 2). For PRKD1 sequencing, fifteen patients tumors were sequenced for Ser910 and in eight samples it was possible to analyze the result. No pathogenic variant was found. The data obtained on PRKD1 in COSMIC were: from 2773 samples, in only 15 (0.54%) mutations were identified in PRKD1, 46% (7/15) came from women aged over 55 years and had tumor molecular subtype Luminal. PRKD1 shows higher mutation frequency in cancer of the large intestine (4.22%) and skin (4.02%). The survival curves constructed in KM Plotter demonstrate the high expression of the gene seems to have a positive impact on the patients survival . Despite the low frequency of mutations in PRKD1 gene, other data demonstrate that it appears to play a role of tumor suppressor gene in breast cancer, which must be inhibited by other mechanisms such as DNA methylation

INTRODUÇÃO: O carcinoma basocelular (CBC) é o tipo de câncer cutâneo mais comum no ser humano. O aparecimento de CBC na maioria das vezes se dá de forma esporádica em indivíduos que se expõem cronicamente ao sol. Eventualmente pode estar associado a síndromes, como: Bazex-Dupré- Christol, Rombo e Gorlin-Goltz. Diferente do que ocorre nas síndromes, os casos de CBCs múltiplos familiares não sindrômicos(CBCMFNS) são poucos estudados, tendo na literatura somente cinco relatos de famílias com a doença. O fenótipo é de múltiplos CBCs superficiais sem presença de outras anormalidades. Devido os CBCs esporádicos e os CBCs presentes na Síndrome de Gorlin-Goltz apresentarem mutações no gene PTCH1, possivelmente os CBCs múltiplos também estejam associados a alterações neste gene. Este gene esta localizado na região 9q22.3 possuindo 23 éxons, tem um papel importante na formação embrionária e de supressão tumoral. OBJETIVO: Análise genética dos éxons 9,11, 16, 17 e 23 do PTCH1 de oito componentes da mesma família, pertencentes a três diferentes gerações, sendo três portadores de CBCs múltiplos, e dentre estes dois suspeitos de CBCMFNS. MÉTODOS: Extração de DNA dos leucócitos do sangue periférico; PCR; clonagem dos produtos de amplificação (pGEM T Easy Vector) e seqüenciamento (Big Dye Terminator Kit). As mutações e polimorfismos encontrados foram comparados com a literatura e banco de dados de mutação do gene PTCH1 (www.cybergene.se/PATCH). RESULTADOS: Duas novas mutações foram encontradas nos pacientes suspeitos de CBCMFNS: uma frameshift nt4130(del C) e uma missense nt4261(A->G). Nos familiares foram encontradas cinco novas mutações: Em um primeiro indivíduo uma missense nt1420(G->T); em um segundo a mesma missense nt1420(G->T) e mais uma missense nt2873(C->T); em um terceiro duas frameshift nt1443 (ins T) e nt1468 (ins T), em dois outros indivíduos, irmãos, uma outra mutação missense nt4130(C->T). Foram encontradas ainda dezoito mutações, não descritas anteriormente, nos íntrons 10,15,16 e 17, algumas se repetindo em todos os indivíduos analisados. CONCLUSÃO: Pela primeira vez estão sendo descritas mutações em éxons e íntrons do gene PTCH1 em indivíduos portadores de CBCMFNS e em alguns de seus familiares. / INTRODUCTION: Basal cell carcinomas (BCC) are the most usual skin cancer that affects human beings. Sporadic BCCs are prevalent, often arising in people chronically exposed to UV radiation from the sun. Eventually BCCs may be associated to different syndroms like Bazex-Dupré-Christol, Rambo and Gorlin. Contrarily to syndromic BCCs, the cases of multiple familiar nonsydromic BCCs(MFNSBCC) have only few studies found in the literature. Only five families have been described to date with the disease. Since sporadic and Gorlin BCCs are associated to many mutations in the PTCH1 gene, we hypothesized that the multiple BCCs phenotype is also associated with mutations in this same gene. The PTCH1 tumor suppressor gene is located in the 9q22.3 chromosomal region, contains 23 exons, and has an important role in embryogenesis. OBJETIVE: To perform genetic analysis of PTCH1 exons 9, 11, 16, 17 e 23. METHODS: Eight individuals belonging to different generations from the same family were studied. Three of them bore multiple BCCs, and two of those were suspect to have MFNSBCC. DNA was extracted from blood leukocytes, submitted to PCR, and the PCR products were cloned (pGEM T Easy Vector, Promega) and sequenced (Big Dye Terminator Kit; ABI Prism 3100 sequencer; Applied Biosystems). The polymorphisms and mutations found were analyzed and compared to literature and PTCH1database (www.cybergene.se/PTCH/). RESULTS: In the patients suspect of MFNSBCC were found two new mutation: one frameshift nt4130(del C) and one missense nt4261(A->G). In the relatives were found five new mutation: Three missense nt1420(G->T); nt2873(C->T); nt4130(C->T); and two frameshift nt1443 (ins T) and nt1468 (ins T). In the introns 10,15,16 and 17 were found eighteen new mutations that were not previously reported. CONCLUSION: For the first time mutation in exons and introns of PTCH1 gene have been described in patients bore MFNSBCC and some of their relatives.

INTRODUÇÃO: O carcinoma basocelular (CBC) é o tipo de câncer cutâneo mais comum no ser humano. O aparecimento de CBC na maioria das vezes se dá de forma esporádica em indivíduos que se expõem cronicamente ao sol. Eventualmente pode estar associado a síndromes, como: Bazex-Dupré- Christol, Rombo e Gorlin-Goltz. Diferente do que ocorre nas síndromes, os casos de CBCs múltiplos familiares não sindrômicos(CBCMFNS) são poucos estudados, tendo na literatura somente cinco relatos de famílias com a doença. O fenótipo é de múltiplos CBCs superficiais sem presença de outras anormalidades. Devido os CBCs esporádicos e os CBCs presentes na Síndrome de Gorlin-Goltz apresentarem mutações no gene PTCH1, possivelmente os CBCs múltiplos também estejam associados a alterações neste gene. Este gene esta localizado na região 9q22.3 possuindo 23 éxons, tem um papel importante na formação embrionária e de supressão tumoral. OBJETIVO: Análise genética dos éxons 9,11, 16, 17 e 23 do PTCH1 de oito componentes da mesma família, pertencentes a três diferentes gerações, sendo três portadores de CBCs múltiplos, e dentre estes dois suspeitos de CBCMFNS. MÉTODOS: Extração de DNA dos leucócitos do sangue periférico; PCR; clonagem dos produtos de amplificação (pGEM T Easy Vector) e seqüenciamento (Big Dye Terminator Kit). As mutações e polimorfismos encontrados foram comparados com a literatura e banco de dados de mutação do gene PTCH1 (www.cybergene.se/PATCH). RESULTADOS: Duas novas mutações foram encontradas nos pacientes suspeitos de CBCMFNS: uma frameshift nt4130(del C) e uma missense nt4261(A->G). Nos familiares foram encontradas cinco novas mutações: Em um primeiro indivíduo uma missense nt1420(G->T); em um segundo a mesma missense nt1420(G->T) e mais uma missense nt2873(C->T); em um terceiro duas frameshift nt1443 (ins T) e nt1468 (ins T), em dois outros indivíduos, irmãos, uma outra mutação missense nt4130(C->T). Foram encontradas ainda dezoito mutações, não descritas anteriormente, nos íntrons 10,15,16 e 17, algumas se repetindo em todos os indivíduos analisados. CONCLUSÃO: Pela primeira vez estão sendo descritas mutações em éxons e íntrons do gene PTCH1 em indivíduos portadores de CBCMFNS e em alguns de seus familiares. / INTRODUCTION: Basal cell carcinomas (BCC) are the most usual skin cancer that affects human beings. Sporadic BCCs are prevalent, often arising in people chronically exposed to UV radiation from the sun. Eventually BCCs may be associated to different syndroms like Bazex-Dupré-Christol, Rambo and Gorlin. Contrarily to syndromic BCCs, the cases of multiple familiar nonsydromic BCCs(MFNSBCC) have only few studies found in the literature. Only five families have been described to date with the disease. Since sporadic and Gorlin BCCs are associated to many mutations in the PTCH1 gene, we hypothesized that the multiple BCCs phenotype is also associated with mutations in this same gene. The PTCH1 tumor suppressor gene is located in the 9q22.3 chromosomal region, contains 23 exons, and has an important role in embryogenesis. OBJETIVE: To perform genetic analysis of PTCH1 exons 9, 11, 16, 17 e 23. METHODS: Eight individuals belonging to different generations from the same family were studied. Three of them bore multiple BCCs, and two of those were suspect to have MFNSBCC. DNA was extracted from blood leukocytes, submitted to PCR, and the PCR products were cloned (pGEM T Easy Vector, Promega) and sequenced (Big Dye Terminator Kit; ABI Prism 3100 sequencer; Applied Biosystems). The polymorphisms and mutations found were analyzed and compared to literature and PTCH1database (www.cybergene.se/PTCH/). RESULTS: In the patients suspect of MFNSBCC were found two new mutation: one frameshift nt4130(del C) and one missense nt4261(A->G). In the relatives were found five new mutation: Three missense nt1420(G->T); nt2873(C->T); nt4130(C->T); and two frameshift nt1443 (ins T) and nt1468 (ins T). In the introns 10,15,16 and 17 were found eighteen new mutations that were not previously reported. CONCLUSION: For the first time mutation in exons and introns of PTCH1 gene have been described in patients bore MFNSBCC and some of their relatives.