2 resultados para Mutação (Biologia) Teses
em RUN (Repositório da Universidade Nova de Lisboa) - FCT (Faculdade de Cienecias e Technologia), Universidade Nova de Lisboa (UNL), Portugal
Resumo:
RESUMO:O glicosilfosfatidilinositol (GPI) um complexo glicolipdico utlizado por dezenas de protenas, o qual medeia a sua ancoragem superfcie da clula. Protenas de superfcie celular ancoradas a GPI apresentam vrias funes essenciais para a manuteno celular. A deficincia na sntese de GPI o que caracteriza principalmente a deficincia hereditria em GPI, um grupo de doenas autossmicas raras que resultam de mutaes nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensveis para a biossntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a ligao do factor de transcrio (FT) Sp1 sua sequncia de reconhecimento, impondo a compactao da cromatina, associada hipoacetilao de histonas, e consequentemente, impedindo a transcrio de PIGM. Desta forma, a adio da primeira manose ao GPI comprometida, a sntese de GPI diminui assim como as protenas ligadas a GPI superficie das clulas. Pacientes com Deficincia Hereditria em GPI-associada a PIGM apresentam trombose e epilesia, e ausncia de hemlise intravascular e anemia, sendo que estas duas ltimas caractersticas definem a Hemoglobinria Paroxstica Nocturna (HPN), uma doena rara causada por mutaes no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficincia em GPI varia entre clulas do mesmo tecido e entre clulas de tecidos diferentes. Por exemplo nos granulcitos e linfcitos B a deficincia em GPI muito acentuada mas nos linfcitos T, fibroblastos, plaquetas e eritrcitos aproximadamente normal, da a ausncia de hemlise intravascular. Os eventos transcricionais que esto na base da expresso diferencial da ncora GPI nas clulas hematopoiticas so desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os nveis de PIGM mRNA variam entre clulas primrias hematopoiticas normais. Adicionalmente, a configurao dos nucleossomas no promotor de PIGM mais compacta em clulas B do que em clulas eritrides e tal est correlacionado com os nveis de expresso de PIGM, isto , inferior nas clulas B. A presena de vrios motivos de ligao para o FT especfico da linhagem megacarioctica-eritride GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrio. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expresso de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrio restritamente eritride, regula a transcrio de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigao do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrio de PIGM em ambas as clulas B e eritride. Curiosamente, ao contrrio do que acontece nas clulas B, em que a transcrio de PIGM requer a ligao do FT geral Sp1 ao motivo -270GC, nas clulas eritrides Sp1 regula a transcrio de PIGM ao ligar-se a montante e no ao motivo -270GC. Para alm disso, demonstrou-se que Sp2 no um regulador directo da transcrio de PIGM quer nas clulas B quer nas clulas eritrides. Estes resultados explicam a ausncia de hemlise intravascular nos doentes com IGD associada a PIGM, uma das principais caractersticas que define a HPN. Por ltimo, resultados preliminares mostraram que a represso da transcrio de PIGM devida mutação patognica -270C>G est associada com a diminuio da frequncia de interaces genmicas em cis entre PIGM e os seus genes vizinhos, sugerindo adicionalmente que a regulao de PIGM e desses genes partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, especfico-detecido, por meio de FTs genricos e especficos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.
Resumo:
RESUMO: Introduo: Tratamento do carcinoma da mama Este trabalho inicia-se com a histria do tratamento do carcinoma da mama, desde os primeiros documentos que descrevem doentes com carcinoma da mama at 1950. Desde 1950 at 2000 o diagnstico, risco e as modalidades teraputicas usadas no tratamento das doentes so mais detalhadas com nfase nas teraputicas locais, regionais e sistmicas. Parte 1:Quem tratar com teraputica sistmica adjuvante Captulo 1: A classificao TNM no est morta no carcinoma da mama Tem sido dito que a classificao TNM no adequada para usar como ferramenta de prognstico e deciso teraputica no carcinoma da mama, especialmente em doentes com carcinoma detectado atravs de rastreio, que tem geralmente menores dimenses. A razo desta classificao no ser adequada prendese com o facto de no estarem incluidos parmetros biolgicos na classificao TNM atual. Pusemos a hiptese de que numa populao com alta percentagem de carcinoma da mama no detectado em exames de rastreio, com uma mediana de idade baixa e com alta percentagem de estadios II e III, o estadiamento clssico, pela classificao TNM, mais descriminatrio que as caractersticas biolgicas na determinao do prognstico. Para isto analismos uma populao de doentes com carcinoma da mama tratados consecutivamente na mesma instituio, durante 10 anos. Caracterizmos os fatores de prognstico do estadiamento clssico includos na classificao TNM e as variantes biolgicas, presentemente no includas na classificao TNM. Quantificmos a capacidade de cada um dos factores de prognstico para para prever a sobrevivncia. A populao de 1699 doentes com carcinoma da mama que foram tratdos com teraputica sistmica adjuvante. Individualmente, cada um dos fatores de prognostico, clssicos ou biolgicos, diferem significativamente entre doentes que sobrevivem e que no sobrevivem. Explicitamente, como previsto, doentes com tumores maiores, envolvimento dos gnglios axilares, estadios TNM mais avanados, que no expressam recetor de esrogneo, com amplificao do gene Her2, triplos negativos ou de menor diferenciao tm menor sobrevida. Na anlise multivariada, s os fatores de prognostico da classificao TNM, o grau histolgico e a amplificao do gene Her2, esta ltima com menos significncia estatistica so preditores independentes de sobrevivncia. Captulo 2: Em busca de novos factores de prognostico: Poder preditivo e mecanismo das alteraes de centrossomas em carcinoma da mama Compilmos inmeros grupos de experincias de genmica feitas em tumores primrios de doentes com carcinoma da mama para as quais existe informao prognstica. Estas experincias so feitas com o objectivo de descobrir novos factores de prognstico. Reanalismos os dados, repetindo a mesma pergunta: Quais so os genes com expresso diferencial estatisticamente significativa entre doentes que recaram e doentes que no recaram. Identificmos 65 genes nestas condies e o MKI67, o gene que codifica a proteina Ki67, estava nesse grupo. Identificmos vrios genes que se sabe estarem envolvidos no processo de agregao de centrossomas. O gene que considermos mais promissor foi a kinesina KiFC1, que j tinha sido identificada como regulador da agregao de centrossomas. Anomalias cetrossomais numricas e estruturais tm sido observadas em neoplasias. H dados correlacionando anolmalias centrossomais estruturais e e numricas com o grau de malignidade e os eventos precoces da carcinognese. Mas estas anomalias centrossomais tm um peso para a clula que deve adapatar-se ou entrar em apoptose. Os nossos resultados sugerem que existe um mecanismo adaptativo, a agregao de centrossomas, com impacto prognstico negativo. O nosso objetivo foi quantificar o valor prognstico das anomalias centrossomais no carcinoma da mama. Para isto usmos material de doentes dos quais sabemos a histria natural. Avalimos os genes de agregao de centrossomas, KIFC1 e TACC3, nas amostras tumorais arquivadas em parafina: primeiro com PCR (polymerase chain reaction) quantitativa e depois com imunohistoqumica (IHQ). Apenas a protena KIFC1 foi discriminatria em IHQ, no se tendo conseguido otimizar o anticorpo da TACC3. Os nveis proteicos de KIFC1 correlacionam-se com mau prognstico. Nas doentes que recaram observmos, no tumor primrio, maior abundncia desta protena com localizao nuclear. Em seguida, demonstrmos que a agregao de centrossomas um fenmeno que ocorre in vivo. Identificmos centrossomas agregados em amostras de tumores primrios de doentes que recaram. Tecnicamente usmos microscopia de fluorescncia e IHQ contra protenas centrossomais que avalimos nos tumores primrios arquivados em blocos de parafina. Observmos agregao de centrossomas num pequeno nmero de doentes que recaram, no validmos, ainda, este fentipo celular em larga escala. Parte 2: Como tratar com teraputica sistmica os vrios subtipos de carcinoma da mama Captulo 3: Quantas doenas esto englobadas na definio carcinoma da mama triplo negativo? (reviso) O carcinoma da mama triplo negativo um tumor que no expressa trs protenas: recetor de estrognio, recetor de progesterona e o recetor do fator de crescimento epidermico tipo 2 (Her2). As doentes com estes tumores no so ainda tratadas com teraputica dirigida, possivelmente porque esta definio negativa no tem ajudado. Sabemos apenas as alteraes genticas que estes tumores no tm, no as que eles tm. Talvez por esta razo, estes tumores so o subtipo mais agressivo de carcinoma da mama. No entanto, na prtica clnica observamos que estas doentes no tm sempre mau prognstico, alm de que dados de histopatologia e epidemiologia sugerem que esta definio negativa no est a capturar um nico subtipo de carcinoma da mama, mas vrios. Avalimos criticamente esta evidncia, clnica, histopatolgica, epidemiolgica e molecular. H evidncia de heterogeneidade, mas no claro quantos subtipos esto englobados nesta definio de carcinoma da mama triplo negativo. A resposta a esta pergunta, e a identificao do fundamento molecular desta heterogeneidade vai ajudar a melhor definir o prognstico e eventualmente a definir novos alvos teraputicos nesta populao difcil. Captulo 4: Terapuica sistmica em carcinoma da mama triplo negativo (reviso) A quimioterapia a nica teraputica sistmica disponvel para as doentes com carcinoma da mama triplo negativo, ao contrrio dos outros dois subtipo de carcinoma da mama que tm com a teraputica antiestrognica e anti Her2, importantes benefcios. Apesar de terem surgido vrias opes teraputicas para estes doentes nennhuma teraputica dirigida foi validada pelos ensaios clnicos conduzidos, possivelmente porque a biologia deste carcinoma ainda no foi elucidada. Muitos ensaios demonstram que os tumores triplos negativos beneficiam com quimioterapia e que as mais altas taxas de resposta patolgica completa teraputica neoadjuvante so observadas precisamente nestes tumors. A resposta patolgica completa correlaciona-se com a sobrevivncia. Estamos a estudar regimes adjuvantes especficos para doentes com estes tumors, mas, neste momento, regimes de terceira gerao com taxanos e antraciclinas so os mais promissores. O papel de subgrupos de frmacos especficos, como os sais de platina, mantmse mal definido. Quanto s antraciclinas e taxanos, estes grupos no mostraram beneficio especfico em carcinoma da mama triplo negativo quando comparado com os outros subtipos. Os prprios carcinomas da mama triplos negativos so heterogneos e carcinomas da mama basais triplos negativos com elevada taxa de proliferao e carcinomas da mama triplos negativos surgidos em doentes com mutação germinal BRCA1 podero ser mais sensveis a sais de platino e menos sensveis a taxanos. Como a definio molecular ainda no foi explicada a busca de teraputica dirigida vai continuar. Captulo 5: Ensaio randomizado de fase II do anticorpo monoclonal contra o recetor do fator de crescimento epidrmico tipo 1 combinado com cisplatino versus cisplatino em monoterapia em doentes com carcinoma da mama triplo negativo metastizado O recetor do fator de crescimento epidrmico tipo 1 est sobre expresso nos tumores das doentes com carcinoma da mama triplo negativo metastizado, um subtipo agressivo de carcinoma da mama. Este ensaio investigou a combinao de cetuximab e cisplatino versus cisplatino isolado em doentes deste tipo. Doentes em primeira ou segunda linha de teraputica para doena metastizada foram randomizadas, num sistema de 2 para 1, para receber at 6 ciclos da combinao de cisplatino e cetuximab ou cisplatino isolado. s doentes randomizadas para o brao de monoterapia podiamos, aps progresso, acrescentar cetuximab ou trat-las com cetuximab isolado. O objetivo primrio foi a taxa de resposta global. Os objetivos secundrios foram a sobrevivncia livre de doena, a sobrevivncia global e o perfil de segurana dos frmacos. A populao em anlise foram 115 doentes tratadas com a combinao e 58 doentes tratadas com cisplatino em monoterapia, 31 destas em quem se documentou progresso passaram a ser tratadas com um regime que inclua cetuximab, isolado ou em combinao. A taxa de resposta global foi de 20% no brao da combinaao e de 10% no brao da monoterapia (odds ratio, 2.13). A sobrevivncia livre de doena foi de 3.7 meses no brao da combinao e de 1.5 meses no brao em monoterapia (hazard ratio, 0.67). A sobrevivncia global foi de 12.9 meses no brao da combinao versus 9.4 meses no brao de cisplatino. Conclui-se que, apesar de no ter sido alcanado o objectivo primrio, acrescentar cetuximab, duplica a resposta e prolonga tanto a sobrevivncia livre de doena como a sobrevivncia global. Captulo 6: Bloquear a angiognese para tratar o carcinoma da mama (reviso) A angiognese uma caracterstica que define a neoplasia, porque tumores com mais de 1mm precisam de formar novos vasos para poderem crescer. Desde que se descobriram as molculas que orquestram esta transformao, que se tm procurado desenvolver e testar frmacos que interfiram com este processo. No carcinoma da mama o bevacizumab foi o primeiro frmaco aprovado pela FDA em primeira linha para tratar doena metasttica. Depois foram estudados um grupo de inibidores de tirosina cinase associados aos recetores transmembranares envolvidos na angiognese como o VEGFR, PDGFR, KIT, RET, BRAF e Flt3: sunitinib, sorafenib, pazopanib e axitinib Neste captulo, analisaram-se e resumiram-se os dados dos ensaios clnicos das drogas anti-angiognicas no tratamaneto do carcinoma da mama. Os ensaios de fase III do bevacizumab em carcinoma da mama mostraram uma reduo na progresso de doena de 22 a 52% e aumento da sobrevivncia livre de doena de 1.2 a 5.5 meses mas nunca foi demonstrado prolongamento de sobrevivncia. Os ensaios de fase III em carcinoma da mama adjuvante com bevacizumab so dois e foram ambos negativos. O ensaio de fase III com o inibidor da tirosina cinase, sunitinib foi negativo, enquanto que os ensaios de fase II com os inibidores da tirosina cinase sorafenib e pazopanib melhoraram alguns indicadores de resposta e sobrevivncia. A endostatina foi testada no contexto neoadjuvante com antraciclinas e melhorou a taxa de resposta, mas, mais ensaios so necessrios para estabelecer este frmaco. A maioria dos ensaios clnicos dos agentes antiangiognicos em carcinoma da mama reportaram aumento da taxa de resposta e de sobrevivncia livre de doena mas nunca aumento da sobrevivncia global quando comparado com quimioterapia isolada o que levou ao cepticismo a que assistimos atualmente em relao ao bloqueio da angiognese. Ensaios clnicos selecionados em doentes especficas com objetivos translacionais relacionados com material biolgico colhido, preferefencialmente em diferentes intervalos da teraputica, sero cruciais para o bloqueio da angiognese sobreviver como estratgia teraputica em carcinoma da mama. Captulo 7: A resposta hipoxia medeia a resistncia primria ao sunitinib em carcinoma da mama localmente avanado O sunitinib um frmaco antiangiognico que nunca foi avaliado isolado em doentes com carcinoma da mama no tratadas. O nosso objetivo foi caracaterizar a atividade do sunitinib isolado e em combinao com o docetaxel em carcinoma da mama no tratado, localmente avanado ou opervel, mas de dimenso superior a 2 cm, para compreender os mecanismos de resposta. Doze doentes foram tratadas com duas semanas iniciais de sunitinib seguido de quatro ciclos de combinao de sunitinib e docetaxel. A resposta, a reistncia e a toxicidade foram avaliadas de acordo com parametros clnicos, ressonncia magntica nuclear, tomografia de emisso de positres, histopatologia e perfis de expresso genmica. Detetmos resistncia primria ao sunitinib na janela inicial de duas semanas, evidenciada em quatro doentes que no responderam. data da cirurgia, cinco doentes tinham tumor vivel na mama e axila, quatro tinahm tumor vivel na mama e trs foram retiradas do ensaio. No houve respostas patolgicas completas. A comparao dos perfis de expresso genmica entre os respondedores e os no respondedores, aos quinze dias iniciais, permitiu-nos identificar sobre expresso de VEGF e outras vias angiognicas nos no respondedores. Especificamente, em tumores resistentes ao sunitinib isolado detectmos uma resposta transcricional hipoxia caracterizada por sobre expresso de vrios dos genes alvo do HIF1. Neste ensaio de sunitinib isolado em doentes no tratadas com carcinoma da mama localmente avanado, encontrmos evidncia molecular de resistncia primria ao sunitinib possivelmente mediada por sobre expresso de genes que respondem hipoxia. Parte 3: Quando parar a teraputica sistmica s doentes com carcinoma da mama Captulo 8: Agressividade teraputica ns ltimos trs meses de vida num estudo retrospetivo dum centro nico Inclumos todos os adultos que morreram com tumores slidos na instituio em 2003 e foram tratados com quimioterapia para tratar neoplaias metastizadas. Colhemos dados detalhados relacionados com quimioterapia e toxicidade nos ltimos trs meses de vida a partir do processo clnico. Trezentas e dezanove doentes foram includos, a mediana de idade foi 61 anos. A mediana de sobrevivncia de doena metasttica foi de 11 meses. 66% (211) dos doentes foram tratados com QT nos ltimos 3 meses de vida, 37% foram tratados com QT no limo ms de vida e 21% nas ltimas duas semanas. Nos doentes que foram tratados com QT nos ltimos trs meses de vida, 50% comearam um novo regime teraputico neste perodo e 14% comearam um novo regime no ltimo ms. Identificmos como determinantes de tratamento com QT no fim de vida a idade jovem, o carcinoma da mama, do ovrio e do pncreas. Conclumos que administrmos QT no fim de vida frequentemente e inicimos novos regimes teraputicos no ltimo ms de vida em 14% dos casos. Precisamos de aprofundar este trabalho para compreender se esta atitude agressiva resulta em melhor paliao de sintomas e qualidade de vida no fim de vida dos doentes com neoplasias disseminadas. Captulo 9: O tratamento do carcinoma da mama no fim de vida est a mudar? Quismos caracterizar a modificao da tendncia no uso de QT e de estratgias paliativas no fim de vida das doentes com carcinoma da mama em diferentes instituies e em intervalos de tempo diferentes. Para isto selecionmos doentes que morreram de carcinoma da mama durante 6 anos, entre 2007 e 2012, num hospital geral e comparmos com as doentes que morreram de carcinoma da mama em 2003 num centro oncolgico. Avalimos um total de 232 doentes. O grupo mais recente tem 114 doentes e o grupo anterior tem 118 doentes. Usmos estatstica descritiva para caracterizar QT no fim de vida e o uso de estratgias paliativas. Ambas as coortes so comparveis em termos das caractersticas do carcinoma da mama. Observmos aumento do uso de estatgias paliativas: consulta da dor, consulta de cuidados paliativos e radioterapia paliativa no cuidado das doentes com carcinoma da mama metastizado. Evidencimos aumento do nmero de mortes em servios de cuidados paliativos. No entanto, a QT paliativa continua a ser prolongada at aos ltimos meses de vida, embora tenhamos mostrado uma diminuio desta prtica. Outros indicadores de agressividade como a admisso hospitalar tambm mostraram diminuio. Confirmmos a nossa hiptese de que h maior integrao da medicina paliativa multidisciplinar e menos agressividade na teraputica sistmica das doentes com carcinoma da mama nos ltimos meses de vida. Chapter 10: Porque que os nossos doentes so tratados com quimioterapia at ao fim da vida? (editorial) Este captulo comea por dar o exmeplo duma jovem de 22 anos que viveu trs meses aps comear QT paliatva. Este caso epitomiza a futilidade teraputica e usado como ponto de partida para explorar as razes pelas quais administramos QT no fim de vida aos doentes quando intil, txica, logisticamente complexa e cara. Ser que estamos a prescrever QT at tarde demais? Os oncologistas fazem previses excessivamente otimistas e tm uma atitude pr teraputica excessiva e so criticados por outros intervenientes nas instituies de sade por isto. Crescentemente doentes, familiares, associaes de doentes, definidores de polticas de sade, jornalistas e a sociedade em geral afloram este tema mas tornam-se inconsistentes quando se trata dum doente prximo em que se modifica o discurso para que se faam teraputicas sitmicas agressivas. H uma crescente cultura de preservao da qualidade de vida, paliao, abordagem sintomtica, referenciao a unidades de cuidados paliativos e outros temas do fim de vida dos doentes oncolgicos terminais. Infelizmente, este tema tem ganhado momentum no porque os oncologistas estejam a refletir criticamente sobre a sua prtica, mas porque os custos dos cuidados de sade so crescentes e incomportveis. Seja qual fr o motivo, as razes que levam os oncologistas a administrar QT no fim de vida devem ser criticamente elucidadas. Mas h poucos dados para nos guiar nesta fase delicada da vida dos doentes e os que existem so por vezes irreconciliveis, uma reviso destes dados que foi feita neste captulo. Concluso: A abordagem do carcinoma da mama no futuro? Na concluso, tenta-se olhar para o futuro e prever como ser a tomada a cargo dum doente com carcioma da mama amanh. Faz-se uma avaliao das vrias reas desde preveno, rastreio, suscetibilidade gentica e comportamental e teraputica. Na teraputica separa-se a teraputica locoregional, sistmica adjuvante e da doena metastizada. Nos trs ltimos pargrafos a histria duma mulher com um carcinoma localmente avanado que sobre expressa o recetor Her2, serve como ilustrao de como devemos estar preparados para incorporar evoluo, heterogeneidade e dinamismo no cuidado de doentes com carcinoma da mama. -------------------------------------------------------------------------------------------------- ABSTRACT: Introduction: Breast cancer care in the past This work starts with an overview of the treatment of breast cancer (BC). From the first reports of patients ill with BC until 1950. From 1950 until 2000, there is a more detailed account on how BC patients were treated with emphasis on the different modalities, local, regional and systemic treatments and their evolution. Part 1: Who to treat with adjuvant systemic therapy? Chapter 1: TNM is not dead in breast cancer It has been said that the current TNM staging system might not be suitable for predicting breast cancer (BC) outcomes and for making therapeutic decisions, especially for patients with screen detected BC which is smaller. The reason for this is also due to the non inclusion of tumor biology parameters in the current TNM system. We hypothesize that in a population where there is still a large abundance of non screen detected BC, with a low median age of incidence and abundance of high TNM staged lesions, biology is still second to classical staging in predicting prognosis. We analyzed a population of consecutive BC patients from a single institution during ten years. We characterized current established prognostic factors, classical staging variables included in the current TNM staging system and biological variables, currently not included in the TNM system. We quantified the capacity of individual prognostic factors to predict survival. We analyzed a population of 1699 consecutive BC patients. We found that individually both the TNM system prognostic factors and the biological prognostic factors are differing among BC survivors and dead patients in a statistically significant distribution. Explicitly, patients with larger tumors, positive nodes, higher stage lesions, ER negative, HER2 positive, TN or lower differentiation tumors show decreased survival. In the multivariate analysis we can conclude that in a population such as ours classical TNM staging variables, irrespective of tumor biological features, are still the most powerful outcome predictors. Chapter 2: Defining breast cancer prognosis: The predictive power and mechanism of centrosome alterations in breast cancer We performed a systematic analysis of the literature and compiled an extensive data set of gene expression data originated in primary tumours of BC patients with prognostic information. We analysed this data seeking for genes consistently up or down regulated in poor prognosis BC, i.e. that relapsed after initial treatment. In the course this bioinformatics analysis our lab identified 65 genes statistically significant across multiple datasets that can discriminate between relapsed and non-relapsed BC patients. Among the identified genes, we have detected genes such as MKI67, a marker of mitotic activity which is routinely used in the clinic. Unexpectedly, we also discovered several genes found to be involved in centrosome clustering, The most prominent of these is the kinesin KIFC1, also called HSET, and previously identified as regulator of centrosome clustering. Centrosome abnormalities (numerical, structural) have been observed in cancer. Indeed, compelling data has shown that cells from many cancers have multiple and abnormal centrosomes, that are either correlated with tumour malignancy or considered an early tumorigenesis event. However, extra centrosomes come at a cost and cells must be able to handle such abnormalities or otherwise die. Thus our results suggested a new mechanism of breast cancer progression with negative prognostic value. We aimed at quantifying the predictive power of centrosome clustering in BC clinical setting and at detecting this process in BC patient material. We validated the centrosome clustering genes KIFC1 and TACC3 in formalin fixed paraffin embedded (FFPE) BC patient material, using quantitative real-time PCR (RT-qPCR) technology. Our results indicate that the tested KIFC1 has a clear IHC signal (1) and that the protein expression patterns and levels correlate with prognosis, with relapsing patients having increased expression and nuclear localisation of this kinesin (2). Next we were able to show that centrosome clustering does occur in vivo. We identified centrosome amplification and clustering in breast cancer samples, and we established a fluorescence microscopy-based IHC approach by staining FFPE samples with centrosomal markers. Using this approach we have observed centrosome amplification and clustering in a small set of poor prognosis samples. By expanding the number of samples in which we have characterised the number of centrosomes, we were able to confirm our preliminary observation that centrosomes are clustered in relapsed BC. Part 2: How to treat breast cancer subtypes? Chapter 3: How many diseases is triple negative breast cancer? (review) Triple negative breast cancer is a subtype of breast cancer that does not express the estrogen receptor, the progesterone receptor and the epidermal growth factor receptor type 2 (Her2). These tumors are not yet treated with targeted therapies probably because no positive markers have been described to reliably classify them - they are described for what they are not. Perhaps for this reason, they are among the most aggressive of breast carcinomas, albeit with very heterogenous clinical behavior. The clinical observation that these patients do not carry a uniformly dismal prognosis, coupled with data coming from pathology and epidemiology, suggests that this negative definition is not capturing a single clinical entity, but several. We critically evaluate this evidence in this paper, reviewing clinical and epidemiological data, as well as molecular data. There is evidence for heterogeneity, but it is not clear how many diseases are grouped into triple negative breast cancer. Answering this question, and identifying the molecular basis of heterogeneity will help define prognosis and, eventually, the identification of new targeted therapies. Chapter 4: Systemic treatment for triple negative breast cancer (review) Chemotherapy remains the backbone of treatment for triple negative breast cancer (TNBC). Despite the appearance of new targeted and biologic agents there has been no targeted therapy validated for TNBC, possibly because the biology of TNBC has not been conclusively elucidated. Many studies have shown that TNBC derive significant benefit of chemotherapy in the neoadjuvant, adjuvant and metastatic treatment, possibly more benefit than other BC subtypes. Neoadjuvant chemotherapy studies have repeatedly shown higher response rates in TNBC than non-TNBC. Pathologic complete response has been shown to predict improved long term outcomes in BC. Although specific adjuvant regimens for TNBC are under study, third generation chemotherapy regimens utilizing dose dense or metronomic polychemotherapy are among the most effective tools presently available. The role of specific chemotherapy agents, namely platinum salts, in the treatment of TNBC remains undefined. Taxanes and anthracyclines are active in TNBC and remain important agents, but have not shown specific benefit over non-TNBC. TNBC is itself a heterogeneous group in which subgroups like basal like BC defined by higher proliferation and including those TNBC arising in BRCA1 mutation carriers may be more sensitive to platinum agents and relatively less sensitive to taxanes. The molecular characterization of TNBC is lacking and therefore the search for targeted therapy is still ongoing. Chapter 5: Randomized phase II study of the anti-epidermal growth factor receptor monoclonal antibody cetuximab with cisplatin versus cisplatin alone in patients with metastatic triple-negative breast cancer Epidermal growth factor receptor is overexpressed in metastatic triple-negative breast cancers, an aggressive subtype of breast cancer. Our randomized phase II study investigated cisplatin with or without cetuximab in this setting. Patients who had received no more than one previous chemotherapy regimen were randomly assigned on a 2:1 schedule to receive no more than six cycles of cisplatin plus cetuximab or cisplatin alone. Patients receiving cisplatin alone could switch to cisplatin plus cetuximab or cetuximab alone on disease progression. The primary end point was overall response rate (ORR). Secondary end points studied included progressionfree survival (PFS), overall survival (OS), and safety profiles. The full analysis set comprised 115 patients receiving cisplatin plus cetuximab and 58 receiving cisplatin alone; 31 patients whose disease progressed on cisplatin alone switched to cetuximab-containing therapy. The ORR was 20% with cisplatin plus cetuximab and 10% with cisplatin alone (odds ratio, 2.13). Cisplatin plus cetuximab resulted in longer PFS compared with cisplatin alone (median, 3.7 v 1.5 months; hazard ratio, 0.67. Corresponding median OS was 12.9 versus 9.4 months. While the primary study end point was not met, adding cetuximab to cisplatin doubled the ORR and appeared to prolong PFS and OS, warranting further investigation in mTNBC. Chapter 6: Blocking angiogenesis to treat breast cancer (review) Angiogenesis is a hallmark of cancer because tumors larger than 1mm need new vessels to sustain their growth. Since the discovery of the molecular players of this process and some inhibitors, that angiogenesis became a promising therapeutic target. Bevacizumab was the first molecular-targeted antiangiogenic therapy approved by the FDA and is used as first-line therapy in metastatic breast cancer. A second class of approved inhibitors (sunitinib, sorafenib, pazopanib and axitinib) include oral small-molecule tyrosine kinase inhibitors that target vascular endothelial growth factor receptors, platelet-derived growth factor receptors, and other kinases including KIT, Ret, BRAF and Flt-3, but none of these have gained approval to treat breast cancer. This review analyzes and summarizes data from clinical trials of anti-angiogenic agents in the treatment of BC. Phase III trials of bevacizumab in advanced BC have demonstrated a reduction in disease progression (2252%), increased response rates and improvements in progression-free survival of 1.2 to 5.5 months, but no improvements in OS. Bevacizumab phase III trials in early BC have both been negative. Bevacizumab combined with chemotherapy is associated with more adverse events. Phase III trials of the tyrosine kinase inhibitor sunitinib were negative, while randomized phase II trials of sorafenib and pazopanib have improved some outcomes. Endostatin has been tested in neoadjuvant clinical trials in combination with anthracyclinebased chemotherapy in treatment-naive patients and has increased the clinical response rate, but more trials are needed to establish this drug. Most trials of anti-angiogenic agents in BC have reported improved RR and PFS but no increase in OS compared to chemotherapy alone, leading to skepticism towards blocking angiogenesis. Selected trials in selected BC populations with translational endpoints related to harvested tumor tissue and other biological material samples, preferentially at several timepoints, will be crucial if antiangiogenesis is to survive as a strategy to treat BC. Chapter 7: Does hypoxic response mediate primary resistance to sunitinib in untreated locally advanced breast cancer? The antiangiogenic drug sunitinib has never been evaluated as single agent in untreated BC patients. We aimed to characterize the activity of sunitinib, alone and with docetaxel, in untreated locally advanced or operable BC, and, to uncover the mechanisms of response. Twelve patients were treated with an upfront window of sunitinib followed by four cycles of sunitinib plus docetaxel. Response, resistance and toxicity were evaluated according to standard clinical parameters, magnetic resonance imaging, positron emission tomography, pathology characterization and gene expression profiling. We detected primary resistance to sunitinib upfront window in untreated BC, as evidenced by four non-responding patients. At surgery, five patients had viable disease in the breast and axilla, four had viable tumor cells in the breast alone and three were taken off study due to unacceptable toxicity and thus not evaluated. Early functional imaging was useful in predicting response. There were no pathologic complete responses (pCR). Comparison of gene expression profiling tumor data between early responders and non-responders allowed us to identify upregulation of VEGF and angiogenic pathways in non responders. Specifically, in tumors resistant to the single-agent sunitinib we detected a transcriptional response to hypoxia characterized by over-expression of several HIF1 target genes. In this report of single-agent sunitinib treatment of untreated localized BC patients, we found molecular evidence of primary resistance to sunitinib likely mediated by up-regulation of hypoxia responsive genes. Part 3: When to stop systemic treatment of breast cancer patients? Chapter 8: The aggressiveness of cancer care in the last three months of life: a retrospective single centre analysis. All adult patients with solid tumors who died in our hospital in 2003 and received chemotherapy for advanced cancer, were included. Detailed data concerning chemotherapy and toxicity, in the last three months of life, were collected from patients clinical charts. A total of 319 patients were included. Median age was 61 years. Median time from diagnosis of metastatic disease to death was 11 months. The proportion of patients who received chemotherapy in the last three months of life was 66% (n=211), in the last month 37% and in the last two weeks 21%. Among patients who received chemotherapy in the last three months of life, 50% started a new chemotherapy regimen in this period and 14% in the last month. There was an increased probability of receiving chemotherapy in the last three months of life in younger patients and in patients with breast, ovarian and pancreatic carcinomas. There was a large proportion of patients who received chemotherapy in the last three months of life, including initiation of a new regimen within the last 30 days. Thus, further study is needed to evaluate if such aggressive attitude results in better palliation of symptoms at the end of life. Chapter 9: Is breast cancer treatment in the end of life changing? We aimed to characterize the shifting trends in use of anti-cancer chemotherapy and palliative care approaches in the end of life of BC patients in different institutions and times. For this, we selected women that died of BC during six years, from 2007 to 2012, and were treated in a central acute care general hospital and compared it with the BC patients that died in 2003 and were treated in a large cancer center. We analyzed a total of 232 patients: the more recent group has 114 women and the older cohort has 118. We used descriptive statistics to characterize CT in the EoL and use of palliative care resources. Both populations were similar in terms of BC characteristics. We observed more palliative care resources, pain clinic, palliative care teams and palliative radiotherapy, involved in the care of MBC patients and a shift towards more deaths at hospices. Systemic anti cancer treatments continue to be prolonged until very late in patients lives, notwithstanding, we could show a decrease in the use of such treatments. Other indicators of aggressiveness, namely hospital admissions, also show a decrease. We confirmed our hypothesis that there is more integration of multidisciplinary palliative care and less aggressiveness in the treatment of metastatic cancer patients, specifically, use of palliative anti-cancer treatment and hospital admissions. Nonetheless, we use systemic therapy until too late with underutilization of palliative medicine. Chapter 10: Why do our patients get chemotherapy until the end of life? (editorial) The editorial starts with a clinical case of a 21 year old patient that lives three months after starting palliative chemotherapy for the first time, a case that illustrates therapeutic futility at the end of life. Why are we not ceasing chemotherapy when it is useless, toxic, logistically complex and expensive? Are we prescribing chemotherapy until too late in solid tumor patients lives? Medical oncologists have overly optimistic predictions and, excessive, treatment-prone attitude and they are criticized by other health care providers for this. Increasingly, patients, their families, advocacy groups, policy makers, journalists and society at large dwell on this topic, which is a perplexing conundrum, because sometimes they are the ones demanding not to stop aggressive systemic anticancer treatments, when it comes to their loved ones. There is a growing culture of awareness toward preserving quality of life, palliative care, symptom-directed care, hospice referral and end of life issues regarding terminal cancer patients. Sadly, this issue is gaining momentum, not because oncologists are questioning their practice but because health care costs are soaring. Whatever the motive, the reasons for administering chemotherapy at the end of life should be known. There are few and conflicting scientific data to guide treatments in this delicate setting and we review this evidence in this paper. Conclusion: What is the future of breast cancer care? This work ends with a view into the future of BC care. Looking into the different areas from prevention, screening, hereditary BC, local, regional and systemic treatments of adjuvant and metastatic patients. The last three paragraphs are a final comment where the story of a patient with Her2 positive locally advanced breast cancer is used as paradigm of evolution, heterogeneity and dynamism in the management of BC.
