Thyroid hormone status interferes with estrogen target gene expression in breast cancer samples in menopausal women

We investigated thyroid hormone levels in menopausal BrC patients and verified the action of triiodothyronine on genes regulated by estrogen and by triiodothyronine itself in BrC tissues. We selected 15 postmenopausal BrC patients and a control group of 18 postmenopausal women without BrC. We measured serum TPO-AB, TSH, FT4, and estradiol, before and after surgery, and used immunohistochemistry to examine estrogen and progesterone receptors. BrC primary tissue cultures received the following treatments: ethanol, triiodothyronine, triiodothyronine plus 4-hydroxytamoxifen, 4-hydroxytamoxifen, estrogen, or estrogen plus 4-hydroxytamoxifen. Genes regulated by estrogen (TGFA, TGFB1, and PGR) and by triiodothyronine (TNFRSF9, BMP-6, and THRA) in vitro were evaluated. TSH levels in BrC patients did not differ from those of the control group (1.34 ± 0.60 versus 2.41 ± 1.10  μ U/mL), but FT4 levels of BrC patients were statistically higher than controls (1.78 ± 0.20 versus 0.95 ± 0.16 ng/dL). TGFA was upregulated and downregulated after estrogen and triiodothyronine treatment, respectively. Triiodothyronine increased PGR expression; however 4-hydroxytamoxifen did not block triiodothyronine action on PGR expression. 4-Hydroxytamoxifen, alone or associated with triiodothyronine, modulated gene expression of TNFRSF9, BMP-6, and THRA, similar to triiodothyronine treatment. Thus, our work highlights the importance of thyroid hormone status evaluation and its ability to interfere with estrogen target gene expression in BrC samples in menopausal women.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T(3)), 17 beta-estradiol (E(2)), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T(3); dish 3: T(3)+TAM; dish 4: TAM; dish 5: E(2); dish 6: E(2)+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T(3) for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T(3) than E(2). Concomitant treatment with TAM had a mitigating effect on the T(3) effect, while E(2) induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E(2).. Endocrinol. Invest. 31: 1047-1051, 2008) (c) 2008, Editrice Kurtis

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family Hp1(Hs alpha), Hp1(Hs beta) and Hp1(Hs gamma) which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1(Hs alpha), HP1(Hs beta) and HP1(Hs gamma) was determined by real-time RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow. (Int J Biol Markers 2008; 23: 219-24)

Clinical decisions in breast cancer, a heterogenous disease

RESUMO: Introdução: Tratamento do carcinoma da mama Este trabalho inicia-se com a história do tratamento do carcinoma da mama, desde os primeiros documentos que descrevem doentes com carcinoma da mama até 1950. Desde 1950 até 2000 o diagnóstico, risco e as modalidades terapêuticas usadas no tratamento das doentes são mais detalhadas com ênfase nas terapêuticas locais, regionais e sistémicas. Parte 1:Quem tratar com terapêutica sistémica adjuvante Capítulo 1: A classificação TNM não está morta no carcinoma da mama Tem sido dito que a classificação TNM não é adequada para usar como ferramenta de prognóstico e decisão terapêutica no carcinoma da mama, especialmente em doentes com carcinoma detectado através de rastreio, que tem geralmente menores dimensões. A razão desta classificação não ser adequada prendese com o facto de não estarem incluidos parâmetros biológicos na classificação TNM atual. Pusemos a hipótese de que numa população com alta percentagem de carcinoma da mama não detectado em exames de rastreio, com uma mediana de idade baixa e com alta percentagem de estadios II e III, o estadiamento clássico, pela classificação TNM, é mais descriminatório que as características biológicas na determinação do prognóstico. Para isto analisámos uma população de doentes com carcinoma da mama tratados consecutivamente na mesma instituição, durante 10 anos. Caracterizámos os fatores de prognóstico do estadiamento clássico incluídos na classificação TNM e as variantes biológicas, presentemente não incluídas na classificação TNM. Quantificámos a capacidade de cada um dos factores de prognóstico para para prever a sobrevivência. A população é de 1699 doentes com carcinoma da mama que foram tratádos com terapêutica sistémica adjuvante. Individualmente, cada um dos fatores de prognostico, clássicos ou biológicos, diferem significativamente entre doentes que sobrevivem e que não sobrevivem. Explicitamente, como previsto, doentes com tumores maiores, envolvimento dos gânglios axilares, estadios TNM mais avançados, que não expressam recetor de esrogéneo, com amplificação do gene Her2, triplos negativos ou de menor diferenciação têm menor sobrevida. Na análise multivariada, só os fatores de prognostico da classificação TNM, o grau histológico e a amplificação do gene Her2, esta última com menos significância estatistica são preditores independentes de sobrevivência. Capítulo 2: Em busca de novos factores de prognostico: Poder preditivo e mecanismo das alterações de centrossomas em carcinoma da mama Compilámos inúmeros grupos de experiências de genómica feitas em tumores primários de doentes com carcinoma da mama para as quais existe informação prognóstica. Estas experiências são feitas com o objectivo de descobrir novos factores de prognóstico. Reanalisámos os dados, repetindo a mesma pergunta: Quais são os genes com expressão diferencial estatisticamente significativa entre doentes que recaíram e doentes que não recaíram. Identificámos 65 genes nestas condições e o MKI67, o gene que codifica a proteina Ki67, estava nesse grupo. Identificámos vários genes que se sabe estarem envolvidos no processo de agregação de centrossomas. O gene que considerámos mais promissor foi a kinesina KiFC1, que já tinha sido identificada como regulador da agregação de centrossomas. Anomalias cetrossomais numéricas e estruturais têm sido observadas em neoplasias. Há dados correlacionando anolmalias centrossomais estruturais e e numéricas com o grau de malignidade e os eventos precoces da carcinogénese. Mas estas anomalias centrossomais têm um peso para a célula que deve adapatar-se ou entrará em apoptose. Os nossos resultados sugerem que existe um mecanismo adaptativo, a agregação de centrossomas, com impacto prognóstico negativo. O nosso objetivo foi quantificar o valor prognóstico das anomalias centrossomais no carcinoma da mama. Para isto usámos material de doentes dos quais sabemos a história natural. Avaliámos os genes de agregação de centrossomas, KIFC1 e TACC3, nas amostras tumorais arquivadas em parafina: primeiro com PCR (polymerase chain reaction) quantitativa e depois com imunohistoquímica (IHQ). Apenas a proteína KIFC1 foi discriminatória em IHQ, não se tendo conseguido otimizar o anticorpo da TACC3. Os níveis proteicos de KIFC1 correlacionam-se com mau prognóstico. Nas doentes que recaíram observámos, no tumor primário, maior abundância desta proteína com localização nuclear. Em seguida, demonstrámos que a agregação de centrossomas é um fenómeno que ocorre in vivo. Identificámos centrossomas agregados em amostras de tumores primários de doentes que recaíram. Tecnicamente usámos microscopia de fluorescência e IHQ contra proteínas centrossomais que avaliámos nos tumores primários arquivados em blocos de parafina. Observámos agregação de centrossomas num pequeno número de doentes que recaíram, não validámos, ainda, este fenótipo celular em larga escala. Parte 2: Como tratar com terapêutica sistémica os vários subtipos de carcinoma da mama Capítulo 3: Quantas doenças estão englobadas na definição carcinoma da mama triplo negativo? (revisão) O carcinoma da mama triplo negativo é um tumor que não expressa três proteínas: recetor de estrogénio, recetor de progesterona e o recetor do fator de crescimento epidermico tipo 2 (Her2). As doentes com estes tumores não são ainda tratadas com terapêutica dirigida, possivelmente porque esta definição negativa não tem ajudado. Sabemos apenas as alterações genéticas que estes tumores não têm, não as que eles têm. Talvez por esta razão, estes tumores são o subtipo mais agressivo de carcinoma da mama. No entanto, na prática clínica observamos que estas doentes não têm sempre mau prognóstico, além de que dados de histopatologia e epidemiologia sugerem que esta definição negativa não está a capturar um único subtipo de carcinoma da mama, mas vários. Avaliámos criticamente esta evidência, clínica, histopatológica, epidemiológica e molecular. Há evidência de heterogeneidade, mas não é claro quantos subtipos estão englobados nesta definição de carcinoma da mama triplo negativo. A resposta a esta pergunta, e a identificação do fundamento molecular desta heterogeneidade vai ajudar a melhor definir o prognóstico e eventualmente a definir novos alvos terapêuticos nesta população difícil. Capítulo 4: Terapêuica sistémica em carcinoma da mama triplo negativo (revisão) A quimioterapia é a única terapêutica sistémica disponível para as doentes com carcinoma da mama triplo negativo, ao contrário dos outros dois subtipo de carcinoma da mama que têm com a terapêutica antiestrogénica e anti Her2, importantes benefícios. Apesar de terem surgido várias opções terapêuticas para estes doentes nennhuma terapêutica dirigida foi validada pelos ensaios clínicos conduzidos, possivelmente porque a biologia deste carcinoma ainda não foi elucidada. Muitos ensaios demonstram que os tumores triplos negativos beneficiam com quimioterapia e que as mais altas taxas de resposta patológica completa à terapêutica neoadjuvante são observadas precisamente nestes tumors. A resposta patológica completa correlaciona-se com a sobrevivência. Estamos a estudar regimes adjuvantes específicos para doentes com estes tumors, mas, neste momento, regimes de terceira geração com taxanos e antraciclinas são os mais promissores. O papel de subgrupos de fármacos específicos, como os sais de platina, mantémse mal definido. Quanto às antraciclinas e taxanos, estes grupos não mostraram beneficio específico em carcinoma da mama triplo negativo quando comparado com os outros subtipos. Os próprios carcinomas da mama triplos negativos são heterogéneos e carcinomas da mama basais triplos negativos com elevada taxa de proliferação e carcinomas da mama triplos negativos surgidos em doentes com mutação germinal BRCA1 poderão ser mais sensíveis a sais de platino e menos sensíveis a taxanos. Como a definição molecular ainda não foi explicada a busca de terapêutica dirigida vai continuar. Capítulo 5: Ensaio randomizado de fase II do anticorpo monoclonal contra o recetor do fator de crescimento epidérmico tipo 1 combinado com cisplatino versus cisplatino em monoterapia em doentes com carcinoma da mama triplo negativo metastizado O recetor do fator de crescimento epidérmico tipo 1 está sobre expresso nos tumores das doentes com carcinoma da mama triplo negativo metastizado, um subtipo agressivo de carcinoma da mama. Este ensaio investigou a combinação de cetuximab e cisplatino versus cisplatino isolado em doentes deste tipo. Doentes em primeira ou segunda linha de terapêutica para doença metastizada foram randomizadas, num sistema de 2 para 1, para receber até 6 ciclos da combinação de cisplatino e cetuximab ou cisplatino isolado. Às doentes randomizadas para o braço de monoterapia podiamos, após progressão, acrescentar cetuximab ou tratá-las com cetuximab isolado. O objetivo primário foi a taxa de resposta global. Os objetivos secundários foram a sobrevivência livre de doença, a sobrevivência global e o perfil de segurança dos fármacos. A população em análise foram 115 doentes tratadas com a combinação e 58 doentes tratadas com cisplatino em monoterapia, 31 destas em quem se documentou progressão passaram a ser tratadas com um regime que incluía cetuximab, isolado ou em combinação. A taxa de resposta global foi de 20% no braço da combinaçao e de 10% no braço da monoterapia (odds ratio, 2.13). A sobrevivência livre de doença foi de 3.7 meses no braço da combinação e de 1.5 meses no braço em monoterapia (hazard ratio, 0.67). A sobrevivência global foi de 12.9 meses no braço da combinação versus 9.4 meses no braço de cisplatino. Conclui-se que, apesar de não ter sido alcançado o objectivo primário, acrescentar cetuximab, duplica a resposta e prolonga tanto a sobrevivência livre de doença como a sobrevivência global. Capítulo 6: Bloquear a angiogénese para tratar o carcinoma da mama (revisão) A angiogénese é uma característica que define a neoplasia, porque tumores com mais de 1mm precisam de formar novos vasos para poderem crescer. Desde que se descobriram as moléculas que orquestram esta transformação, que se têm procurado desenvolver e testar fármacos que interfiram com este processo. No carcinoma da mama o bevacizumab foi o primeiro fármaco aprovado pela FDA em primeira linha para tratar doença metastática. Depois foram estudados um grupo de inibidores de tirosina cinase associados aos recetores transmembranares envolvidos na angiogénese como o VEGFR, PDGFR, KIT, RET, BRAF e Flt3: sunitinib, sorafenib, pazopanib e axitinib Neste capítulo, analisaram-se e resumiram-se os dados dos ensaios clínicos das drogas anti-angiogénicas no tratamaneto do carcinoma da mama. Os ensaios de fase III do bevacizumab em carcinoma da mama mostraram uma redução na progressão de doença de 22 a 52% e aumento da sobrevivência livre de doença de 1.2 a 5.5 meses mas nunca foi demonstrado prolongamento de sobrevivência. Os ensaios de fase III em carcinoma da mama adjuvante com bevacizumab são 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 sobrevivência. A endostatina foi testada no contexto neoadjuvante com antraciclinas e melhorou a taxa de resposta, mas, mais ensaios são necessários para estabelecer este fármaco. A maioria dos ensaios clínicos dos agentes antiangiogénicos em carcinoma da mama reportaram aumento da taxa de resposta e de sobrevivência livre de doença mas nunca aumento da sobrevivência global quando comparado com quimioterapia isolada o que levou ao cepticismo a que assistimos atualmente em relação ao bloqueio da angiogénese. Ensaios clínicos selecionados em doentes específicas com objetivos translacionais relacionados com material biológico colhido, preferefencialmente em diferentes intervalos da terapêutica, serão cruciais para o bloqueio da angiogénese sobreviver como estratégia terapêutica em carcinoma da mama. Capítulo 7: A resposta à hipoxia medeia a resistência primária ao sunitinib em carcinoma da mama localmente avançado O sunitinib é um fármaco antiangiogénico que nunca foi avaliado isolado em doentes com carcinoma da mama não tratadas. O nosso objetivo foi caracaterizar a atividade do sunitinib isolado e em combinação com o docetaxel em carcinoma da mama não tratado, localmente avançado ou operável, mas de dimensão 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 combinação de sunitinib e docetaxel. A resposta, a reistência e a toxicidade foram avaliadas de acordo com parametros clínicos, ressonância magnética nuclear, tomografia de emissão de positrões, histopatologia e perfis de expressão genómica. Detetámos resistência primária ao sunitinib na janela inicial de duas semanas, evidenciada em quatro doentes que não responderam. À data da cirurgia, cinco doentes tinham tumor viável na mama e axila, quatro tinahm tumor viável na mama e três foram retiradas do ensaio. Não houve respostas patológicas completas. A comparação dos perfis de expressão genómica entre os respondedores e os não respondedores, aos quinze dias iniciais, permitiu-nos identificar sobre expressão de VEGF e outras vias angiogénicas nos não respondedores. Especificamente, em tumores resistentes ao sunitinib isolado detectámos uma resposta transcricional à hipoxia caracterizada por sobre expressão de vários dos genes alvo do HIF1α. Neste ensaio de sunitinib isolado em doentes não tratadas com carcinoma da mama localmente avançado, encontrámos evidência molecular de resistência primária ao sunitinib possivelmente mediada por sobre expressão de genes que respondem à hipoxia. Parte 3: Quando parar a terapêutica sistémica às doentes com carcinoma da mama Capítulo 8: Agressividade terapêutica ns últimos três meses de vida num estudo retrospetivo dum centro único Incluímos todos os adultos que morreram com tumores sólidos na instituição em 2003 e foram tratados com quimioterapia para tratar neoplaias metastizadas. Colhemos dados detalhados relacionados com quimioterapia e toxicidade nos últimos três meses de vida a partir do processo clínico. Trezentas e dezanove doentes foram incluídos, a mediana de idade foi 61 anos. A mediana de sobrevivência de doença metastática foi de 11 meses. 66% (211) dos doentes foram tratados com QT nos últimos 3 meses de vida, 37% foram tratados com QT no úlimo mês de vida e 21% nas últimas duas semanas. Nos doentes que foram tratados com QT nos últimos três meses de vida, 50% começaram um novo regime terapêutico neste período e 14% começaram um novo regime no último mês. Identificámos como determinantes de tratamento com QT no fim de vida a idade jovem, o carcinoma da mama, do ovário e do pâncreas. Concluímos que administrámos QT no fim de vida frequentemente e iniciámos novos regimes terapêuticos no último mês de vida em 14% dos casos. Precisamos de aprofundar este trabalho para compreender se esta atitude agressiva resulta em melhor paliação de sintomas e qualidade de vida no fim de vida dos doentes com neoplasias disseminadas. Capítulo 9: O tratamento do carcinoma da mama no fim de vida está a mudar? Quisémos caracterizar a modificação da tendência no uso de QT e de estratégias paliativas no fim de vida das doentes com carcinoma da mama em diferentes instituições e em intervalos de tempo diferentes. Para isto selecionámos doentes que morreram de carcinoma da mama durante 6 anos, entre 2007 e 2012, num hospital geral e comparámos com as doentes que morreram de carcinoma da mama em 2003 num centro oncológico. Avaliámos um total de 232 doentes. O grupo mais recente tem 114 doentes e o grupo anterior tem 118 doentes. Usámos estatística descritiva para caracterizar QT no fim de vida e o uso de estratégias paliativas. Ambas as coortes são comparáveis em termos das características do carcinoma da mama. Observámos aumento do uso de estatégias paliativas: consulta da dor, consulta de cuidados paliativos e radioterapia paliativa no cuidado das doentes com carcinoma da mama metastizado. Evidenciámos aumento do número de mortes em serviços de cuidados paliativos. No entanto, a QT paliativa continua a ser prolongada até aos últimos meses de vida, embora tenhamos mostrado uma diminuição desta prática. Outros indicadores de agressividade como a admissão hospitalar também mostraram diminuição. Confirmámos a nossa hipótese de que há maior integração da medicina paliativa multidisciplinar e menos agressividade na terapêutica sistémica das doentes com carcinoma da mama nos últimos meses de vida. Chapter 10: Porque é que os nossos doentes são tratados com quimioterapia até ao fim da vida? (editorial) Este capítulo começa por dar o exmeplo duma jovem de 22 anos que viveu três meses após começar QT paliatva. Este caso epitomiza a futilidade terapêutica e é usado como ponto de partida para explorar as razões pelas quais administramos QT no fim de vida aos doentes quando é inútil, tóxica, logisticamente complexa e cara. Será que estamos a prescrever QT até tarde demais? Os oncologistas fazem previsões excessivamente otimistas e têm uma atitude pró terapêutica excessiva e são criticados por outros intervenientes nas instituições de saúde por isto. Crescentemente doentes, familiares, associações de doentes, definidores de políticas de saúde, jornalistas e a sociedade em geral afloram este tema mas tornam-se inconsistentes quando se trata dum doente próximo em que se modifica o discurso para que se façam terapêuticas sitémicas agressivas. Há uma crescente cultura de preservação da qualidade de vida, paliação, abordagem sintomática, referenciação a unidades de cuidados paliativos e outros temas do fim de vida dos doentes oncológicos terminais. Infelizmente, este tema tem ganhado momentum não porque os oncologistas estejam a refletir criticamente sobre a sua prática, mas porque os custos dos cuidados de saúde são crescentes e incomportáveis. Seja qual fôr o motivo, as razões que levam os oncologistas a administrar QT no fim de vida devem ser criticamente elucidadas. Mas há poucos dados para nos guiar nesta fase delicada da vida dos doentes e os que existem são por vezes irreconciliáveis, é uma revisão destes dados que foi feita neste capítulo. Conclusão: A abordagem do carcinoma da mama no futuro? Na conclusão, tenta-se olhar para o futuro e prever como será a tomada a cargo dum doente com carcioma da mama amanhã. Faz-se uma avaliação das várias àreas desde prevenção, rastreio, suscetibilidade genética e comportamental e terapêutica. Na terapêutica separa-se a terapêutica locoregional, sistémica adjuvante e da doença metastizada. Nos três últimos parágrafos a história duma mulher com um carcinoma localmente avançado que sobre expressa o recetor Her2, serve como ilustração de como devemos estar preparados para incorporar evolução, 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 (22–52%), 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 HIF1α 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 patientsʼ 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 patientsʼ 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.

Increased expression of urokinase-type plasminogen activator mRNA determines adverse prognosis in ErbB2-positive primary breast cancer.

PURPOSE: To evaluate and validate mRNA expression markers capable of identifying patients with ErbB2-positive breast cancer associated with distant metastasis and reduced survival. PATIENTS AND METHODS: Expression of 60 genes involved in breast cancer biology was assessed by quantitative real-time PCR (qrt-PCR) in 317 primary breast cancer patients and correlated with clinical outcome data. Results were validated subsequently using two previously published and publicly available microarray data sets with different patient populations comprising 295 and 286 breast cancer samples, respectively. RESULTS: Of the 60 genes measured by qrt-PCR, urokinase-type plasminogen activator (uPA or PLAU) mRNA expression was the most significant marker associated with distant metastasis-free survival (MFS) by univariate Cox analysis in patients with ErbB2-positive tumors and an independent factor in multivariate analysis. Subsequent validation in two microarray data sets confirmed the prognostic value of uPA in ErbB2-positive tumors by both univariate and multivariate analysis. uPA mRNA expression was not significantly associated with MFS in ErbB2-negative tumors. Kaplan-Meier analysis showed in all three study populations that patients with ErbB2-positive/uPA-positive tumors exhibited significantly reduced MFS (hazard ratios [HR], 4.3; 95% CI, 1.6 to 11.8; HR, 2.7; 95% CI, 1.2 to 6.2; and, HR, 2.8; 95% CI, 1.1 to 7.1; all P < .02) as compared with the group with ErbB2-positive/uPA-negative tumors who exhibited similar outcome to those with ErbB2-negative tumors, irrespective of uPA status. CONCLUSION: After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.

Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tamoxifen inhibits transforming growth factor-α gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T3), 17β-estradiol (E2), and tamoxifen (TAM) on transforming growth factor (TGF)-α gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T3; dish 3: T3+TAM; dish 4: TAM; dish 5: E2; dish 6: E2+TAM. TGF-α mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T3 for 48 h significantly increased TGF-α mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-α mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-α mRNA expression is more efficiently upregulated by T3 than E2. Concomitant treatment with TAM had a mitigating effect on the T3 effect, while E2 induced TGF-α upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-α, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER α and β; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E2. ©2008, Editrice Kurtis.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Two prognostic factors in breast cancer: Novel functions of EGFR and beta-catenin

In this study, we demonstrated the novel functions of two important prognostic markers in breast cancer, EGFR and b -catenin in proliferation and/or other transformation phenotype. ^ First we demonstrated that EGFR could be detected in the nucleus in highly proliferating tissues, including primary breast cancer samples and a breast cancer cell line. We found that EGFR contained a strong transactivation domain, complexed with an AT-rich consensus DNA sequence and activated promoters containing this sequence, including cyclin D1 promoter. Therefore, EGFR may function as a transcription factor to activate genes required for highly proliferating activity such as cyclin D1 in breast cancer. ^ In the second part of this study, we identified b -catenin as an important prognostic factor in breast cancer. We found that cyclin D1 was one of the genes regulated by b -catenin in breast cancer cells. The transactivation activity of b -catenin correlated significantly with cyclin D1 expression in both breast cancer cell lines and in breast cancer patient samples, in which high b -catenin activity correlated with poor prognosis of the patients. Moreover, blockage of b -catenin activity significantly inhibited transformation phenotypes in breast cancer cells. Therefore, our results indicate that b -catenin can be involved in breast cancer formation and/or progression and may serve as a target for breast cancer therapy. ^

Involvement of lipocalin 2 in leukemia and breast cancer

Human lipocalin 2 is described as the neutrophil gelatinase-associated lipocalin (NGAL). The lipocalin 2 gene encodes a small, secreted glycoprotein that possesses a variety of functions, of which the best characterized function is organic iron binding activity. Elevated NGAL expression has been observed in many human cancers including breast, colorectal, pancreatic and ovarian cancers. I focused on the characterization of NGAL function in chronic myelogenous leukemia (CML) and breast cancer. Using the leukemic xenograft mouse model, we demonstrated that over-expression of NGAL in K562 cells, a leukemic cell line, led to a higher apoptotic rate and an atrophy phenotype in the spleen of inoculated mice compared to K562 cells alone. These results indicate that NGAL plays a primary role in suppressing hematopoiesis by inducing apoptosis within normal hematopoietic cells. In the breast cancer project, we analyzed two microarray data sets of breast cancer cell lines ( n = 54) and primary breast cancer samples (n = 318), and demonstrated that high NGAL expression is significantly correlated with several tumor characteristics, including negative estrogen receptor (ER) status, positive HER2 status, high tumor grade, and lymph node metastasis. Ectopic NGAL expression in non-aggressive (ZR75.1 and MCF7) cells led to aggressive tumor phenotypes in vitro and in vivo. Conversely, knockdown of NGAL expression in various breast cancer cell lines by shRNA lentiviral infection significantly decreased migration, invasion, and metastasis activities of tumor cells both in vitro and in vivo . It has been previously reported that transgenic mice with a mutation in the region of trans-membrane domain (V664E) of HER2 develop mammary tumors that progress to lung metastasis. However, we observed that genetic deletion of the 24p3 gene, a mouse homolog of NGAL, in HER2 transgenic mice by breeding with 24p3-null mice resulted in a significant delay of mammary tumor formation and reduction of lung metastasis. Strikingly, we also found that treatment with affinity purified 24p3 antibodies in the 4T1 breast cancer mice strongly reduced lung metastasis. Our studies provide evidence that NGAL plays a critical role in breast cancer development and progression, and thus NGAL has potential as a new therapeutic target in breast cancer.^

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approx5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11–12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21–q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3–q4 and 18p11.31 and gains of 6p25.1–p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2–11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01

FKBPL and its peptide derivative, AD-01, have already demonstrated well-established inhibitory effects on breast cancer growth and CD44 dependent anti-angiogenic activity1, 2, 3. Since breast cancer stem cells (BCSCs) are CD44 positive, we wanted to explore if AD-01 could specifically target BCSCs. FKBPL stable overexpression or AD-01 treatment were highly effective at reducing the BCSC population measured by inhibiting mammosphere forming efficiency (MFE) in cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- subpopulation, validated these results. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed across three generations of mammospheres, where mammospheres were completely eradicated by the third generation (p<0.001). Clonogenic assays suggested that AD-01 mediated BCSC differentiation, with a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones. In support of this, the stem cell markers, Nanog and Oct4 were significantly reduced following AD-01 treatment, whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in mammosphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). When AD-01 was combined with other agents, we observed synergistic activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Importantly, using ‘gold standard’ in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, AD-01 appears to have dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.