Iron and related proteins in the breast cancer microenvironment: expression patterns in epithelial and stromal inflammatory cells and association with clinicopathological markers of behavior and progression

ORIANA ALEXANDRA DE PAIXÃO PRAÇAS MARQUES IRON AND RELATED PROTEINS IN THE BREAST CANCER MICROENVIRONMENT: EXPRESSION PATTERNS IN EPITHELIAL AND STROMAL INFLAMMATORY CELLS AND ASSOCIATION WITH CLINICOPATHOLOGICAL MARKERS OF BEHAVIOR AND PROGRESSION Tese de Candidatura ao grau de Doutor em Patologia e Genética Molecular submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Professor Doutor Carlos Alberto da Silva Lopes Categoria - Professor Catedrático Jubilado Afiliação - Departamento de Patologia e Imunologia Molecular, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Co-Orientadora – Professora Doutora Maria da Graça Beça Gonçalves Porto Categoria - Professora Catedrática Convidada Afiliação - Departamento de Patologia e Imunologia Molecular, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Co-Orientadora – Professora Doutora Maria Berta de Jesus Duarte da Silva Categoria - Professora Associada Afiliação - Departamento de Patologia e Imunologia Molecular, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Financial Support: PhD fellowship: SFRH/BD/78184/2011 Fundação para a Ciência e Tecnologia (FCT) v DIRETIVAS LEGAIS Ao abrigo do disposto do nº 2, alínea a) do artigo 31º do Decreto-Lei n.º 230/2009 declara-se que a autora desta dissertação contribuiu activamente na conceptualização, execução, interpretação e escrita dos seguintes manuscritos aceites, submetidos e em preparação: O. Marques, B. Martins da Silva, G. Porto, C. Lopes, Iron Homeostasis in Breast Cancer, Cancer Lett. 347 (2014) 1-14 O. Marques, G. Porto, A. Rêma, F. Faria, A. Cruz Paula, M. Gomez-Lázaro, P. Silva, B. Martins da Silva, C. Lopes, Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment, BMC Cancer 16 (2016) 187200 A. Rosa, O. Marques, L. Leite, A. Cruz Paula, A. Rêma, P. Faustino, B. Martins da Silva, C. Lopes, G. Porto, CCL2 Expression in Breast Ductal Carcinomas: a novel modulator of local iron homeostasis? (Submitted for Publication) O. Marques, A. Rosa, L. Leite, P. Faustino, A. Rêma, B. Martins da Silva, G. Porto, C. Lopes, HFE Variants and the Expression of Iron Related Proteins in Breast Cancer (In Preparation) O. Marques, A. Canadas, F. Faria, E. Oliveira, I. Amorim, F. Seixas, A. Gama, A. Lobo-da-Cunha, B. Martins da Silva, G. Porto, C. Lopes, Expression of Iron-related Proteins in Feline and Canine Mammary Gland Reveals Unexpected Accumulation of Iron (Submitted for Publication) This thesis includes unpublished results. vii AGRADECIMENTOS Embora corra o risco de fazer uma lista interminável de agradecimentos, é impossível finalizar este trabalho sem agradecer profundamente às pessoas que para ele contribuíram. Esta tese reflecte não só o meu trabalho, mas também o contributo individual daqueles que estiveram ao meu lado, pessoal e profissionalmente. A todos os que me ajudaram a chegar ao final deste caminho, o meu mais sincero obrigado. Ao Professor Doutor Carlos Lopes, na qualidade de Orientador desta tese, que me acompanha desde 2008, o meu maior agradecimento pela orientação científica e por me ter guiado desde o início. Por ter acreditado em mim, mas também por me fazer ver a realidade quando a minha imaginação teimava em dar asas. Não poderia deixar de mencionar que a admiração que tenho pelo Professor ultrapassa a relação profissional, e que a sua forma de ser perante o próximo, me fez também crescer. À Professora Doutora Graça Porto, co-Orientadora desta tese, por ser a minha ‘companheira’ incansável nesta jornada. Por ter estado disponível e ao meu lado every step of the way e me mostrar a beleza da biologia do ferro. Por acreditar tanto ou mais do que eu. Por me abrir o caminho para o crescimento pessoal e profissional. Não tenho palavras para exprimir o quanto agradecida estou e o carinho que desenvolvi pela Professora nos últimos 5 anos. À Professora Doutora Berta Martins da Silva, co-Orientadora desta tese, por ter acreditado neste trabalho e ter aceitado que ele fosse desenvolvido no laboratório por si dirigido. Muito obrigada por todas as críticas construtivas e ajuda na resolução de problemas. À Professora Doutora Fátima Gärtner pela disponibilidade e por ter permitido que pudesse fazer parte deste trabalho com amostras biológicas do Laboratório de Patologia Veterinária do Instituto de Ciências Biomédicas Abel Salazar. À Professora Doutora Maria de Sousa por ter tido amabilidade de me guiar na publicação do primeiro artigo com resultados originais apresentado nesta tese e por exigir que desse o melhor de mim. À Doutora Paula Faustino por me ter recebido de braços abertos no Laboratório de Biologia Molecular do Centro de Genética Humana (Instituto Nacional de Saúde Ricardo Jorge), e me ter dado condições para fazer a genotipagem do HFE. viii À Professora Doutora Fátima Carneiro por tão amavelmente ter permitido que utilizasse o microscópio de microdissecção a laser no Serviço de Anatomia Patológica do Hospital de São João, e à Doutora Paula Silva por ter sido a colega das horas intermináveis na sua utilização. Às Doutoras Maria Gomez-Lázaro e Maria José Oliveira, por partilharem da paixão por ciência e contribuírem activamente com ideias para este trabalho. À Dra. Rita Sampaio do Serviço de Anatomia Patológica do Hospital de Santo António pela ajuda na organização dos dados clinico-patológicos das doentes incluídas neste estudo. A todos os elementos actuais e passados do Laboratório de Imunogenética, do Instituto de Ciências Biomédicas Abel Salazar, Sandra, São, Andreia, Ana Fonseca e Ana Tavares, Bárbara, Cláudia, Daniela e Joana, por terem estado ao meu lado nesta caminhada, pela discussão científica e disponibilidade para ajudar em todas as alturas. Um agradecimento especial à Ana Rosa e Luciana Leite, que estiveram directamente envolvidas neste trabalho no ano da sua conclusão, por me terem ajudado não só ao nível técnico e laboratorial, mas também por me terem ajudado a crescer enquanto pessoa. À Alexandra Rêma e Fátima Carvalho do Laboratório de Patologia Veterinária do Instituto de Ciências Biomédicas Abel Salazar, por terem sido incansáveis na ajuda e passagem de conhecimento ao nível das técnicas de histopatologia. Mas acima de tudo, por terem sido sempre um ombro amigo. À Ana Canadas e Irina Amorim, pela amizade, acompanhamento e disponibilidade para discussões científicas em torno da patologia veterinária. Aos antigos colegas BCRIB (Basic and Clinical Research on Iron Biology) por todas as discussões científicas extremamente enriquecedoras e por todas as críticas construtivas a este trabalho. Ao Professor Doutor Alexandre Lobo da Cunha e Elsa Oliveira do Laboratório de Biologia Celular do Instituto de Ciências Biomédicas Abel Salazar pela ajuda preciosa ajuda com a Microscopia Electrónica e cooperação científica no trabalho de patologia veterinária comparativa. A todos os meus amigos. Àqueles que estiveram presentes nos ‘bons dias para a Ciência’ mas que também não abandonaram o barco nos maus momentos. Sou deveras uma felizarda por ter como amigos pessoas tão boas que acreditam em mim. No entanto, não posso deixar de fazer um agradecimento especial a determinadas pessoas que ix estiveram constantemente a puxar-me para a tona ou que tiveram um papel preponderante em algum ponto do decorrer desta tese. Ao Arnaud, que nunca se recusou a ajudar-me e que será o meu eterno companheiro desta jornada (e de outras passadas). À Inês, Luísa Margarida, Rui, Xana e Xanoca, verdadeiros representantes da amizade incondicional, sempre com as atitudes e palavras certas para me motivar. Ao Zé, Gabi, César e o ‘nosso’ Moonshine Pub, por providenciarem os momentos de descontracção entre amigos tão necessários. À Maria Luísa e Ricardo, que embora amigos de curta data, me acolheram e apoiaram como se fosse sua ‘filha’. Ao Gonçalo, que para além de ter sido um crítico revisor do inglês de grande parte desta tese, esteve persistentemente ao meu lado durante a escrita desta, facilitando a tarefa de uma forma que acredito que nem ele imagina. E, por fim, à pessoa mais importante no meu mundo: a minha Mãe. Aquela que acredita em mim incondicionalmente e a quem devo a pessoa em que me tornei. xvi (invasive ductal carcinomas) for hepcidin (n= 121), FPN1 (Ferroportin 1, n= 113), TFR1 (Transferrin Receptor 1, n= 119) and FT (Ferritin, n= 119); (B). Immunoexpression score for macrophages in control normal samples, DCIS (ductal carcinomas in situ) and IDC (invasive ductal carcinomas) for Hepcidin (n= 75), FPN1 (Ferroportin 1, n= 62), TFR1 (Transferrin Receptor 1, n= 73) and FT (Ferritin, n= 91); (C). Immunoexpression score for lymphocytes in control normal samples, DCIS (ductal carcinomas in situ) and IDC (invasive ductal carcinomas) for Hepcidin (n= 77), FPN1 (Ferroportin 1, n= 70), TFR1 (Transferrin Receptor 1, n= 73) and FT (Ferritin, n= 91). ..................................................................................................................................................... 78 Figure 3. FPN1-expressing leukocytes are composed by a mixture of CD4 and CD8 T-cells. Sections of normal breast tissue, DCIS (ductal carcinoma in situ) and IDC (invasive ductal carcinoma) to reveal the presence of CD4 and CD8 T cells, in FPN1-expressing leukocyte infiltrate. For details see Materials and Methods (Original magnification ×100upper FPN images, ×400CD4 and CD8 images). ............................................................................................................... 78 Figure 4. FPN1-expressing leukocytes in carcinomas are predominantly M2-like.Sections of normal breast tissue, DCIS (ductal carcinoma in situ) and IDC (invasive ductal carcinoma) to reveal the presence of cells of the macrophage lineage (CD68), and its classical polarization phenotypes, M1-like (CD80) and M2-like (CD163), in FPN1-expressing leukocyte infiltrate. For details see Materials and Methods (Original magnification ×100upper CD68 images, ×400squared image series). ......................................................................................................................................... 79 Figure 5. Breast cancer tissue presents a higher accumulation of iron than normal breast. (A) Percentage of normal and breast cancer samples presenting hemosiderin deposits in epithelial and stromal inflammatory cells; (B-C) Representative images of Perls’ iron staining of a normal (B) and DCIS (C) sample, showing pronounced deposition of hemosiderin in stromal inflammatory cells (arrows) and to a lesser extent in ductal epithelial cells (asterisk) (Original magnification×200, ×400). ......................................................................................................................................... 79 Figure 6. Hepcidin/FPN1 and TFR1/FT phenotype dyads in ductal carcinoma in situ lesions (DCIS). A semi-quantitative method of assessing the immunoexpression in the TMA sections was applied by multiplying the area and intensity staining scores, as described in materials and methods. The scores ranged from 0 to 15. Graphs show Mean ± SEM. Significant differences are shown for comparison with the precedent group *p< 0.05, **p< 0.01, ***p< 0.001, Mann Whitney’s U test; (A). Immunoexpression score for breast epithelial cells in DCIS pure lesions and DCIS lesions in IDC for Hepcidin (n= 35), FPN1 (Ferroportin 1, n= 35), TFR1 (Transferrin Receptor 1, n= 32) and FT (Ferritin, n= 36); (B). Immunoexpression score for macrophages in DCIS pure lesions and DCIS lesions in IDC for Hepcidin (n= 28), FPN1 (Ferroportin 1, n= 27), TFR1 (Transferrin Receptor 1, n= 30) and FT (Ferritin, n= 33); (C). Immunoexpression score for lymphocytes in DCIS pure lesions and DCIS lesions in IDC for Hepcidin (n= 26), FPN1 (Ferroportin 1, n= 31), TFR1 (Transferrin Receptor 1, n= 30) and FT (Ferritin, n= 33). ........................................................... 80 xvii Figure 7. Representative images of the FPN1 analysis by Imaging Flow Cytometry in breast cancer core biopsies. Epithelial cells (EC) are stained by an anti-cytokeratin (CK) FITC. T lymphocytes (T Ly) were identifiable by CD3 PerCP-Cy5.5, B lymphocytes (B Ly) by CD20 PE-Cy7 and macrophages (M0) by CD68 PE-Cy7 (Original magnification ×400). ................................. 81 Figure 8. Representative images of Hepcidin, FPN1, TFR1 and FT immunostaining in nonmetastized and metastized lymph nodes. Archived lymph nodes from cases with previously analyzed primary invasive ductal carcinomas were sectioned and subjected to immunohistochemistry, as described in materials and methods. Boxes indicate lymph node areas near metastasis. Note prominent germinal centers in non-metastized lymph nodes (×400 original magnification figures are shown below its ×200 correspondent figures). M, metastasis; LN, lymph node. ............................................................................................................................................ 82 Chapter 4 Figures Figure 1. Evidences of hemosiderin deposition in a DCIS lesion (a) and in an IDC lesion (b). DABenhanced Perls’ staining of breast tumors, particularly in epithelial (a) and stromal inflammatory cells (b and c). Original magnification of 200X (a and b) and 400X (inset). ................................ 96 Figure 2. Presence of hemosiderin deposition in pure lesions of the breast, in epithelial (grey) and in stromal inflammatory cells (black). Chi-square test for iron deposition in stromal inflammatory cells in pure lesions: between normal and pure DCIS p=0.011; between normal and pure IDC p=0.001 (*p< 0.05, **p< 0.01, ***p< 0.001). Abbreviations: EC, epithelial cells; SIC, stromal inflammatory cells; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; EC – epithelial cells; SIC – stromal cells. ............................................................................................................ 97 Figure 3. Presence of hemosiderin deposition in the tissue adjacent to breast lesions, in epithelial (grey) and in stromal inflammatory cells (black). Chi-square test for iron deposition in stromal inflammatory cells: p=0.011 (*p< 0.05, **p< 0.01, ***p< 0.001). Abbreviations: EC, epithelial cells; SIC, stromal inflammatory cells; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma. ..................................................................................................................................................... 98 Figure 4. Median epithelial CCL2 expression in (a) pure lesions of the breast and (b) in nonneoplastic tissue adjacent to breast lesions. (a) Dunn-Bonferroni test: between normal and pure IDC lesions p=0.007. Error bars: 95% CI. (*p< 0.05, **p< 0.01, ***p< 0.001). Abbreviations: DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma. .......................................................... 99 Figure 5. Infiltration of CCL2-positive macrophages in (a) pure lesions and (b) in non-neoplastic tissue adjacent to breast lesions. (a) Dunn-Bonferroni test: between normal and pure DCIS p<0.001; between normal and pure IDC p=0.001. Error bars: 95% CI. (*p< 0.05, **p< 0.01, ***p< 0.001, versus precedent group) Abbreviations: DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma. ................................................................................................................................. 100 xviii Figure 6. Proposed mechanism of CCL2-induced pathway: CCL2 (green circles), produced by breast tumor cells, attract circulating CCR2-positive cells, such as macrophages and lymphocytes, which are iron-loaded. When these stromal inflammatory cells arrive to the tumor microenvironment, FPN 1 expression is upregulated, partially by the expression of CCL2, and iron (Fe) is supplied for proliferating epithelial cells. Iron supplied by macrophages and lymphocytes leads to increased proliferation and CCL2 secretion by tumor cells, triggering the paracrine signalling pathway, between tumor and immune cells. Macrophages are also accountable for the production of CCL2, resulting in increased leukocyte infiltration and increased iron supply for proliferating neoplastic cells. ..................................................................................................... 106 Chapter 5 Figures Figure 1. Iron-related proteins in invasive breast carcinomas according to HFE genotype. Median hepcidin (A), FPN1 (B), TFR1 (C) and FT (D) expression, in epithelial cells, lymphocytes and macrophages, in relation to the presence or absence (WT) of the p.C282Y and p.H63D variants. Scores ranged from 0 to 15 and errors bars present 95% CI. Abbreviations: WT, Wild-Type; FPN1, ferroportin 1; TFR1, transferrin receptor 1; FT, ferritin. ............................................................. 118 Figure 2. Iron deposition in invasive breast carcinomas according to the HFE genotype. Percentage of invasive breast cancer cases presenting iron deposition in epithelial (black) and stromal inflammatory (grey) cells, in relation to the HFE genotype. Abbreviations: WT, Wild-Type ................................................................................................................................................... 120 Chapter 6 Figures Figure 1. Hepcidin, FPN1, TFR1 and FT expression pattern in the mammary gland tissue of cats and dogs. Representative images of the Hepcidin, FPN1 (Ferroportin 1), TFR1 (Transferrin Receptor 1) and FT (Ferritin) immunostaining in benign and malignant breast lesions. Tissue microarrays containing several samples of cats’ and dogs’ mammary tissue were constructed, sectioned and subjected to immunohistochemistry, as described in materials and methods (Scale bar 50 µm; Scale bar in inset 25 µm). ....................................................................................... 136 Figure 2. Iron accumulation in the mammary tissue of cats and dogs. The percentage of cases presenting hemosiderin deposition, as assessed by Perls’ Prussian Blue staining, in epithelial and stromal inflammatory cells from benign and malignant mammary lesions from cats and dogs is shown. ....................................................................................................................................... 139 Figure 3. Light (A) and electron microscopy (B-C) detection of iron by Perls’ Prussian Blue staining in a normal canine mammary gland. (A) Positive reaction for hemosiderin in macrophages (arrows) and in small granules (arrowheads) located close to the nucleus (nu) in epithelial cells of mammary ducts (asterisks) (Scale bar 50 µm; Scale bar in inset 10 µm); (B) Unstained ultrathin section showing highly electron-dense Prussian blue deposits (arrowheads) within siderosomes close to xix the nucleus (nu) in an epithelial cell (Scale bar 0.5 µm); (C) Stained ultrathin section also revealing iron deposits (arrowheads) within siderosome (Scale bar 0.5 µm); (D) Siderosome (asterisk) in a stained ultrathin section of a control sample without Perls' reaction (Scale bar 0.2 µm) .......... 140 xxi TABLES INDEX Chapter 1 Tables Table 1. Origin sites for common breast diseases........................................................................ 5 Table 2. Breast Cancer Risk Factors. ........................................................................................... 8 Chapter 3 Tables Table 1. Number of spots included in TMA receiver blocks ....................................................... 75 Table 2. FPN1 median expression in IDC samples assessed by Imaging Flow Cytometry ....... 82 Table 3. Analysis of iron-related proteins expression in the LN of patients with IDC ................. 83 Table 4. FPN1 expression in epithelial cells and macrophages in carcinomas according to clinicopathological variables ........................................................................................................ 83 Table 5. Correlation table between tumor size and TFR1 expression ........................................ 84 Chapter 5 Tables Table 1. Expression of iron-related proteins in HFE p.C282Y/p.H63D compound heterozygous IDC patients is increased in comparison with patients without the p.C282Y variant. ...................... 119 Table 2. Clinicopathological features of breast cancer patients according to their HFE genotype. ................................................................................................................................................... 121 Chapter 6 Tables Table 1. Immunoexpression of iron-related proteins in mammary gland tissue ....................... 135 Table 2. Correlation table between grade and the expression of iron-related proteins ............ 138 Supplementary Table 1. Immunoexpression of iron-related proteins in feline mammary gland tissue, per type of lesion. ........................................................................................................... 148 Supplementary Table 2. Immunoexpression of iron-related proteins in canine mammary gland tissue, per type of lesion. ........................................................................................................... 149 xxiii BOXES INDEX Chapter 1 Boxes Box 1. Most common breast cancer histological subtypes, based on the World Health Organization classification of breast tumors ............................................................................... 11 xxv ABBREVIATIONS ADH – Atypical Ductal Hyperplasia ALAS - AminoLevulinic Acid Synthase APES - 3-AminoPropyltriEthoxySilane Arg1 – Arginase 1 ASR – Age-Standardized Rate Bcrp – Breast cancer resistance protein BCS – Breast-Conserving Surgery Bmp - Bone morphogenetic protein BpT - 2-Benzoylpyridine Thiosemicarbazone BRCA – BReast CAncer genes 1 and 2 CAF - Cancer-Associated Fibroblast CCL - Chemokine (C-C motif) Ligand CCL2 - Chemokine (C-C motif) Ligand 2 CCR2 - C-C Chemokine Receptor type 2 CD – Cluster of Differentiation CDC – Cell Division Cycle CHO – Chinese Hamster Ovary ChT – ChemoTherapy CI – Confidence Interval CISH - Chromogenic In Situ Hybridization CK – CytoKeratin COX - CycloOXygenase Cp - Ceruloplasmin CSF-1 - Colony Stimulating Factor 1 CSF1R - Colony Stimulating Factor 1 Receptor CXCL - C-X-C chemokine Ligand DAB - DiAminoBenzidine DCIS – Ductal Carcinoma In Situ Dcyt B - Duodenal cytochrome B D-exo - Desferri-exochelin DFO – DesFerriOxamine DMBA - 7,12-DiMethylBenz(a)Anthracene DMT - divalent metal transporter DpT - Di-2-pyridylketone Thiosemicarbazone e.g. – exampli gratia ECM – ExtraCellular Matrix EGF - Epidermal Growth Factor EGFR - Epidermal Growth Factor Receptor EMT - Epithelial-to-Mesenchymal Transition ER – Estrogen Receptor ERE – Estrogen Responsive Element ERK - Extracellular signal-Regulated Kinase ET – Endocrine Therapy FEA - Flat Epithelial Atypia Fe-S – iron-sulfur FFPE - Formalin-Fixed Paraffin-Embedded FGF2 - Fibroblast Growth Factor 2 FGFR2 - Fibroblast Growth Factor Receptor 2 xxxii pelo facto que, comparativamente com amostras de mamoplastias estéticas de redução, uma maior percentagem de casos de carcinoma da mama apresenta acumulação de ferro em células inflamatórias do estroma. Adicionalmente, alterações na expressão destas proteínas reguladoras do metabolismo celular do ferro estão associadas com o status dos receptores hormonais e tamanho do tumor na altura do diagnóstico. O CCL2 é uma citocina pertencente à família das quimiocinas C-C com um papel comprovado como recrutadora de leucócitos para o microambiente tumoral. Recentemente foi descrito que esta também seria capaz de modelar os níveis locais de ferro no tecido. A expressão de CCL2 nas células epiteliais, previamente descrita como aumentada em casos de carcinoma da mama e confirmado no nosso estudo, foi demonstrada aqui ser também significativamente superior nos casos que apresentam deposição de ferro nas células inflamatórias do estroma. De facto, para além da expectável associação com a infiltração de linfócitos e macrófagos CCL2-positivos, a expressão de CCL2 está também correlacionada com a expressão de ferroportina 1 nos linfócitos. Estas relações sugerem a existência de uma via de sinalização parácrina onde o CCL2 pode ter um papel indirecto na regulação da nutrição e progressão tumoral. Os níveis de expressão de CCL2 no tecido mamário não são significativamente diferentes em doentes com as variantes p.C282Y e/ou p.H63D do gene HFE. No entanto, doentes heterozigóticas compostas p.C282Y/p.H63D apresentam uma maior expressão de hepcidina em linfócitos e macrófagos do infiltrado inflamatório, bem como de receptor da transferrina 1 nos tipos celulares analisados. Surpreendentemente, a expressão das proteínas reguladoras do metabolismo do ferro não é significativamente diferente entre lesões mamárias benignas e malignas em amostras de gatas e cadelas. A observação de que as células tumorais mamárias de gatas e cadelas não apresentam maiores exigências de ferro do que as suas congéneres benignas pode ser explicada pelo facto de que a glândula mamária normal destas espécies apresenta já uma forte acumulação de ferro, ao contrário do que se verifica na espécie humana. Em conclusão, os nossos resultados reforçam a necessidade de estudar o microambiente tumoral no carcinoma da mama, enquanto ampliam o conhecimento sobre a contribuição das células do sistema imune para a regulação local do ferro, em contexto tumoral. Adicionalmente, a translação do estudo original em animais modelo de carcinoma da mama permitiu-nos detectar diferenças fisiológicas nas mamas das gatas e cadelas em relação à mama da mulher, levantando a necessidade de um conhecimento mais aprofundado sobre a regulação sistémica e local da homeostasia do ferro nestas espécies. Chapter 1 General Introduction Chapter 1. General Introduction – Breast Cancer 3 1.1. Breast Cancer 1.1.1. The Human Breast The breast is a dynamic organ that suffers several changes during the embryonic development, puberty, pregnancy, lactation and involution. The development of the ductal system is termed branching morphogenesis, and although it starts in the fetus, it is only completed during puberty when hormonal stimulation triggers differentiation [1, 2]. Under hormonal influence, complex reciprocal interactions between epithelial and stromal cells drive the alterations observed in the mammary gland in a woman’s lifetime. The adult female breast lies on the anterior chest wall, over the pectoralis major muscle, and is placed normally between the 2nd and 6th ribs of the chest wall in the vertical axis and between the sterna edge and the midaxillary line in the horizontal axis [3, 4]. The breast also extends superolaterally to the axilla [5]. The breast is composed by 15-20 lobes, each one drained by a collecting duct. These collecting ducts link the nipple with lactiferous sinus, that by its turn are connected to terminal duct-lobular units (TDLUs), the breast functional unit, by lactiferous and major ducts. Lobules consist of TDLUs, acini and their underlying, hormone-responsive, supporting stroma, consisting of various proportions of fibrous and adipose tissue [6] (Figure 1). Except for a part of the collecting ducts near the nipple, composed of squamous epithelium, the ductal system consists of two main epithelial layers: an inner ductal layer composed of columnar luminal epithelial cells and an outer spindle-shaped myoepithelial cell layer. The epithelial cells of the inner layer are typically immunoreactive for low molecular weight cytokeratins (CK) 8, 18 and 19 and characterized by a cytoplasm with abundant organelles involved in secretion. The outer layer is characterized by the expression of high molecular weight CKs 5/6, anti-actin antibodies and p63, in close contact with the basal lamina [3, 4]. Myoepithelial cells are responsible not only by the maintenance of the ductal structure but also by assisting milk ejection [7]. The basal lamina in conjunction with the epithelial-myoepithelial cell layers and the surrounding zone of delimitating fibroblasts is denominated of epithelial-stromal junction [3]. Lymphocytes, plasma cells and macrophages normally lie in the interstices of this fibroblast network, which facilitates cell-cell interactions [8]. The intralobular stroma is more cellular and contains more vessels than the interlobular stroma, while the latter is more collagenized [3, 4]. The complex lymphatic network present in the mammary gland is drained, mostly, Chapter 1. General Introduction – Breast Cancer 4 to the axillary lymph nodes, which facilitates epithelial cell spread in the case of metastatic disease [9]. 1.1.2. Breast Cancer Breast female lesions are frequent. As fear of cancer is very common, use of image technology in screening is generalized and identifies most of the lesions, when no clinical manifestation still exists. If present, they manifest as palpable nodules or masses [12]. Although most of known epithelial benign and malignant lesions of the breast arise at the level of the TDLU other common breast diseases also have origin in other sites [13](Table 1). Figure 1. A. Schematic representation of the main anatomic features in an adult woman’s breast. Adapted from [10]. B-D. Mammary gland characteristic histological elements, highlighting the ducts, connective and adipose tissue, lobules and acini. Adapted from [2, 11]. Chapter 1. General Introduction – Breast Cancer 5 Table 1. Origin sites for common breast diseases. Adapted from [13]. Nipple  Paget’s Disease  Nipple Adenoma Lactiferous Ducts  Subsclerosing Duct Hyperplasia  Duct Ectasia Segmental and Subsegmental Ducts  Solitary Intraductal Papilloma  Duct Ectasia Terminal Duct Lobular Units  Cyst  Epithelial Hyperplasia  Noninvasive and Invasive Carcinoma Fibrocystic changes are the most common alterations in the adult premenopausal women and thought to be caused by breast alterations during menstruation cycles [12]. Although of little clinical significance, some benign changes may confer an increased risk for the development of breast cancer [14-16]. Even though the exact mechanisms involved in the development of breast cancer remain unknown, the most commonly accepted model hypothesizes that invasive cancer starts in the TDLU [17] and progresses through pre-malignant breast disease by increasing cellular anomalies leading to exaggerated proliferation and atypia [18-20]. A proliferative growth advantage in flat epithelial atypia (FEA) can then give rise to atypical ductal hyperplasia (ADH) that may progress to ductal carcinoma in situ (DCIS) upon additional molecular alterations [21, 22]. Several epidemiologic studies support this stepwise model of progression in which invasiveness arises through the accumulation of abnormalities in benign breast diseases [14, 16, 23, 24]. Each stage is considered to result from genetic alterations in a transformed clonal lineage, eventually capable of invasion and metastasis [25]. Furthermore, matched genetic and epigenetic alterations are frequently found in proliferative diseases without atypia (PDWA), ADH, DCIS and invasive breast cancer (IBC) in the same breast [18, 26-28], supporting a sequential relation between precursor lesions and the IBC with which they are associated [25]. Surprisingly, morphologically normal terminal duct lobules adjacent to breast cancer may already present loss of heterozygosity (LOH) in genes critical for early progression of tumorigenesis [29]. However, the exact place and how DCIS progresses to IBC remains a matter of discussion, with two theories attempting to explain it (Figure 2). The theory of linear progression states that low-grade DCIS evolves linearly to high-grade DCIS and this Chapter 1. General Introduction – Breast Cancer 6 is the entity accountable for the progression to IBC [15, 30-32]. On the other hand, the theory of parallel disease hypothesizes that low-grade DCIS progresses to low-grade IBC, while high-grade DCIS evolves to high-grade IBC, implying a commitment of a particular type of DCIS to its IBC counterpart [33]. This model is supported by cytogenetic studies showing that specific patterns of chromosomal alterations in particular grades of DCIS correspond to distinct genomic changes in matching IBCs [34, 35] and that the degree of DCIS differentiation was correlated with that of the corresponding IBC [36]. However, the concept that these two models are mutually exclusive may underestimate the complexity of the process [37]. Figure 2. Stepwise model of breast cancer development highlighting the two views of progression from ductal carcinoma in situ to invasive breast carcinoma (Theory of Linear Progression and Theory of Parallel Disease). Abbreviations: FEA, flat epithelial atypia; HUT, hyperplasia of usual type; ADH, atypical ductal hyperplasia; DCIS, ductal carcinoma in situ; DCISlow, low-grade ductal carcinoma in situ; DCIShigh, high-grade ductal carcinoma in situ; IBC, invasive breast cancer; IBClow, low-grade invasive breast cancer; IBChigh, high-grade invasive breast cancer. 1.1.2.1. Epidemiology Besides major efforts for disease prevention and treatment, cancer is still a main public health problem. In 2012, 3.4 million new cancer cases were diagnosed and 1.75 million people died of neoplastic disease worldwide [38]. Breast cancer is the most commonly diagnosed type of cancer, closely followed by colorectal cancer [38, 39]. In women, breast cancer is by far the most frequently diagnosed neoplasm, representing nearly 30% of the total, and ranks as the fifth cause of cancer-related death [38, 39]. Populations at higher risk of incidence and mortality are situated in Northern America, Western and Northern Europe (Figure 3A). Although presenting a slightly higher prevalence than the mean established for European Union countries, Portugal follows the Chapter 1. General Introduction – Breast Cancer 7 tendency, with breast cancer representing the most frequently diagnosed type of cancer and cause of cancer death in women (Figure 3B). In Portugal, in 2012, 6066 new cases of breast cancer were diagnosed, with 1570 women succumbing to the disease [39]. Although breast cancer mortality rates have been decreasing since the 1990’s, specifically due to increasingly efficient screening regimens detecting the disease as early as possible, its high incidence demands a greater awareness and investment in treatment and education. Figure 3. A. Breast cancer age-standardized rate (ASR) of incidence and mortality, per 100000 habitants, in the World. B. Age-standardized rate of incidence and mortality of the top 20 most common types of cancer in Portugal. From [39]. 1.1.2.2. Risk Factors Several factors have been consistently associated with an increased risk for the development of breast cancer. Although the following do not deplete the list of possible influencing factors, they clearly highlight the multifactorial etiology of the disease. Breast cancer risk factors can be divided in two main groups. The first includes inherent factors such as age, gender and family history. The second relates to extrinsic factors influenced by the woman’s lifestyle that may condition the neoplastic context to a certain degree [40]. Next, a table summarizing the main established risk factors for breast cancer is presented. Chapter 1. General Introduction – Breast Cancer 8 Table 2. Breast Cancer Risk Factors. Intrinsic Factors Gender Personal History Genetic Predisposition Age Family History Other Breast Diseases Early Menarche Parity, age of full term pregnancy and breastfeeding Age at menopause Race Testosterone  Breast cancer is predominantly diagnosed in women [41].  Previous history of DCIS, hormone receptor negative IDC and young age [42].  Genes in which mutations increase risk: Breast cancer genes (BRCA) 1 and 2, P53, PTEN, STK11, CDH-1 [43-47].  Risk increases with age [48, 49].  Increased in case of history of affected firstdegree relatives, particularly at young age (<50 y.) [50, 51].  Proliferative diseases, especially with atypia confer a substantial increased risk [15, 52].  2 y. delay in menarche corresponds to a risk reduction of 10% [53].  Nulliparousity, parity at an older age (>35 y.) and absence of breast-feeding increases risk [49, 54, 55].  Every year delay increases the risk by 3% [53, 56].  Higher rate of mortality in Black women, due to higher incidence of triple-negative (TN) tumors [49].  High circulating levels of testosterone in postmenopausal women increase risk [57]. Extrinsic Factors Hormone Replacement Therapy (HRT) Obesity Physical Activity Alcohol Consumption Radiation  Breast cancer risk is higher in hormone replacement therapy (HRT) users, especially when started close to menopause [58, 59].  Elevated risk with increasing weight, body mass index and hip circumference [58, 60].  Physical activity reduces risk in a dosedependent manner [61, 62].  Increased risk with binge drinking [63].  Radiation exposure at young age (<35 y.) increases risk [64-66]. Chapter 1. General Introduction – Breast Cancer 9 1.1.2.3. Carcinoma In Situ Carcinoma in situ is a non-obligate precursor of invasive carcinoma, characterized by an abnormal proliferation of epithelial cells confined in the ductal system by the basement membrane, without invasion of the surrounding stroma [67, 68]. The presence of malignant epithelial cells beyond the basement membrane is routinely recognized as individual cells or irregular nests through the stroma. The disruption of the myoepithelial layer may be demonstrated by the absence of myoepithelial cell markers such as p63, calponin or smooth muscle myosin [69]. Initially, carcinomas in situ were divided into lobular carcinomas in situ (LCIS) and ductal carcinomas in situ based on the assumption that these preinvasive lesions were generated either on lobules or on ducts, respectively. However, studies by Wellings and collaborators revealed that most of the lesions arised in the TDLU, marking the end of the distinction between ductal and lobular subtypes [17, 70, 71], which is merely morphological. The characteristic histological aspect of ‘historic’ LCIS is hallmarked by the absence of E-cadherin expression. However, it is not an exclusive aspect, with DCIS also demonstrating E-cadherin loss [72]. Through the observation of nuclear atypia DCIS can be further classified in low, intermediate and high-grade groups [21, 23, 68]. Historically, the architectural pattern of the DCIS was described and it correlated well with several tumor markers. Categories included non-comedo (cribiform, papillary, micropapillary and papillary) and comedosubtypes [73]. The comedo subtype was characterized by the presence of central ductal necrosis and high-grade cells, and associated with estrogen receptor (ER) negativity, Human Epidermal growth factor Receptor 2 (HER2) amplification, p53 mutations and a high proliferation rate [74-79]. With the increasing number of diagnosed DCIS, became apparent that the classification in comedo/ non-comedo was not adequate to classify, for example lesions with central necrosis and low-grade cell pleomorphism. The need for refinement led to several proposed classification schemes until the one in effect today [80, 81]. Size of the lesion is also an important histological parameter to include in the breast pathology report given its clinical significance [68]. Until the 1980’s DCIS was rarely diagnosed and represented less than 1% of detected breast cancers [82]. However, with the introduction of efficient mammography screening regimens detection of early breast cancer improved significantly. Nowadays, DCIS constitutes 20-25% of newly diagnosed breast cancer in the United States of America [83]. Implementation of screening mammography led to a significant reduction of the average size of DCIS lesion at presentation, diminishing from 60 to 10mm [84]. In spite of a clear increase in the Chapter 1. General Introduction – Breast Cancer 16 According to the aforementioned molecular studies, breast carcinomas can be categorized in the following subtypes:  ER-positive: o Luminal A o Luminal B  ER-negative: o HER-2 o Basal/ Triple-negative o Normal breast-like Luminal A This subgroup is characterized by hormone receptor positivity (ER and PR), HER2 negative status and a low proliferation index and represents of 50-60% of diagnosed invasive carcinomas [178]. They are also positive for luminal cytokeratins (CKs), such as CKs 8/18 and present upregulation of certain estrogen receptor related genes (GATA3, FIXA1 and LIV1) [101]. These tumors are usually well-differentiated, of histological low grade and associated with a better prognosis, lower relapse rates and improved overall survival in comparison with other subtypes [179-183]. Given that this type of tumors presents a high expression of hormone receptors, they are usually treated with endocrine therapy or aromatase inhibitors [101, 102]. Luminal B Luminal B tumors constitute 10-20% of invasive breast carcinomas, and include HER-2 positive tumors. In comparison with Luminal A they present a more variable degree of hormone receptor positivity and a higher cell proliferation rate, as shown by the upregulation of proliferation associated genes, such as CCNB1, MYBL2 and MKI67 [165, 184]. They are commonly of higher grade and biologically more aggressive, exhibiting a worse prognosis with higher chances of relapse than Luminal A tumors [179, 182, 183, 185]. The inclusion of the Ki-67 labeling index brought significant clinical value to the identification of luminal subgroups with poorer prognosis [186]. HER-2 These comprise, approximately, 15% of invasive breast carcinomas and are characterized by HER-2 gene amplification or HER2 protein overexpression [187]. However, part of HER-2 tumors are not clinically HER-2 positive, with patients being treated according to clinical testing results [188]. Additionally, about 40% of tumors in this subgroup present p53 mutations [102]. Consequently, HER-2 positive tumors are Chapter 1. General Introduction – Breast Cancer 17 traditionally associated with an aggressive behavior, multifocal/ multicentric disease and extensive nodal involvement [37]. However, the outcome for these tumors has been improved due to the introduction of anti-HER-2 agents in chemotherapy regimens, such as trastuzumab and lapatinib [189-191]. Basal/ Triple-negative Basal-like tumors present a distinct phenotype, lacking ER, PR and HER-2 expression, but displaying overexpression of basal/ myoepithelial cytokeratins (CKs 5/6, 14 and 17) [192]. Approximately 75% of basal-like tumors exhibit p53 gene mutations [165] and increased EGFR expression [101]. They are more frequent in patients with BRCA1 mutations and particularly of younger age [193]. Histologically, they are generally of high-grade and proliferation index presenting with pushing borders and peritumoral lymphocytic infiltrate. Comparing with luminal tumors, triple-negative carcinomas tend to have a poorer prognosis, with a significantly shorter relapse-free survival, and highly variable response rates to chemotherapy [194]. Treatment for this type of breast tumors remains an active area of research, with the introduction of Poly (ADP-ribose) polymerase (PARP) inhibitors [195, 196] and anti-angiogenesis agents [197, 198] showing promising results. Main therapeutic strategies for the management of breast cancer involve surgery, chemotherapy and radiotherapy. The type and eventual combination of treatments should be decided and provided by a multidisciplinary team specialized in breast cancer [199, 200]. 1.1.3. Breast Tissue Microenvironment 1.1.3.1. Normal Breast Microenvironment The mammary gland is constituted by several cellular types that work in synchrony by engaging in complex heterotypic communications with neighbour cells and the underlying ECM resulting in a coordinated normal development and function. The microenvironment surrounds the bilayered epithelial ducts and is composed by extracellular matrix and stromal cells, including adipocytes, fibroblasts, myofibroblasts, endothelial cells and several types of leukocytes [201-204]. The mammary gland is a particular organ once part of its development occurs post-natally, more specifically during puberty and pregnancy. Branching morphogenesis refers to the complex developmental program that results in ductal infiltration into the Chapter 1. General Introduction – Breast Cancer 18 surrounding stroma as a response to hormonal stimulus [202]. Several studies brought evidence on the fact that cell differentiation and branching architecture is influenced by the tissue microenvironment. Cells grown on plastic culture dishes are not able to differentiate into epithelial cells capable of milk production while cells cultured in three-dimensional reconstituted membrane are [205]. Grafting embryonic salivary gland in adult mammary gland tissue or mammary gland epithelium in salivary stroma results in changed epithelial architecture [206, 207]. Additionally, mammary gland tumor co-culture with embryonic mammary gland stroma leads to tumor differentiation and similar growth to controls [208, 209]. Moreover, a series of studies have demonstrated that culturing tissues from other origin in mammary gland microenvironment causes differentiation into functioning mammary epithelial cells organized in ducts [210-212]. In this context, myoepithelial cells are of particular importance given that they not only influence the differentiation, polarity and proliferation of epithelial cells, as they also, in combination with the basement membrane, constitute a physical barrier between epithelial and stromal cell niches [213, 214]. Signaling through essential basement membrane components, such as laminin-322 and collagen IV are essential for branching architecture given that genetic knockout (KO) of collagen IV receptor (integrin α2β1) subunits blunts branching morphogenesis during pregnancy [215-217]. In turn, laminins are essential for tissue-specific differentiation through several mechanisms, such as polarity definition or cytoskeleton reorganization [202, 218]. Adipocytes constitute one of the microenvironment central players, as they contribute to vascularization, epithelial cell proliferation and leukocyte recruitment through the release of several growth factors and chemokines, besides providing a frame for branching epithelia support [219, 220]. Fibroblasts are also key elements in the mammary gland tissue microenvironment. Its primary function is to participate in the deposition of the collagen-enriched ECM underlying the mammary gland ducts [221, 222]. As other tissue microenvironment cell types, fibroblasts modulate branching morphogenesis by producing factors that act on epithelial cells. Hepatocyte growth factor (HGF) is of particular importance, given that it influences epithelial cell proliferation, migration and branching by activating PI3K signaling [223-226]. Fibroblast growth factor 2 (FGF2) is also necessary for the branching of mammary epithelial cells and has also been shown to promote angiogenesis and consequently contribute to a proper oxygen and nutrient supply [227-229]. In addition to the above mentioned players, several types of leukocytes are also necessary for a correct mammary gland development. For example, macrophages that Chapter 1. General Introduction – Breast Cancer 19 are recruited to the tissue stroma in response to epithelial cell colony stimulating factor-1 (CSF-1) production, not only clear shed epithelial cells from the ducts during lumen formation, but also contribute to the arrangement of fibrillar type I collagen, terminal end bud geometry and side branching [230-232]. Similarly, mast cells and eosinophils, recruited through eotaxin secretion, also participate in branching morphogenesis [232, 233]. These studies point to the presence of immune system cells as a normal aspect of a functional mammary gland tissue. However, recent studies provide evidence that alterations in the number and profile of tumor infiltrating immune cells is associated with poor outcome [234]. 1.1.3.2. Breast Tumor Microenvironment Invasive breast tumors lie in a complex microenvironment composed of an altered ECM and several stromal cell populations, most of which increase in cell number during the carcinogenic process. In fact, not only cell numbers increase in general as all breast tissue cell types present a transformed pattern of gene expression during cancer progression [235-239]. The importance of the microenvironment for invasion has been clearly demonstrated in a study by Ma and colleagues revealing that while 5900 genes are differentially expressed in epithelial cells between normal and DCIS, only three genes are differentially expressed between DCIS and invasive carcinoma [236]. Although pathologists have long noticed that certain histopathological characteristics, such as leukocyte infiltration or fibrosis present prognostic value, only more recently evidences have been provided suggesting that tissue microenvironment heterogeneity contributes to the typical features of breast cancer molecular subtypes [240-243]. Actually, changes in the tissue microenvironment may occur even earlier that at the DCIS stage, with epithelial cell signaling resulting in the secretion of chemokines that cause the accumulation of leukocytes, fibroblasts, myofibroblasts, mesenchymal and endothelial cells (Figure 5) [235, 236, 244-246]. Chapter 1. General Introduction – Breast Cancer 20 Figure 5. Alterations in the tissue microenvironment during breast cancer progression. From [201]. Increased expression of genes associated with invasion and angiogenesis is also observed in myoepithelial cells from DCIS, revealing the potential role of these alterations in the collapse of the basement membrane. In this sense, myoepithelial cells function as “natural tumor supressors” by maintaining the physical basement membrane barrier that is lost during invasion [214, 247-249]. Furthermore, the expression genes associated with myoepithelial cell differentiation (smooth muscle actin [SMA], oxytocin receptor [OXTR]) is also lost or downregulated in DCIS [235].The lack of clonally selected gene mutations in myoepithelial and stromal cells suggests that epigenetic mechanisms may be the potential drivers of altered expression patterns in these cell populations [203, 250-252]. Subsequent paracrine, autocrine, juxtacrine, chemokine and heterotypic cellular communications govern breast cancer progression, similarly as they guide mammary gland morphogenesis [202]. Malignant epithelial cell signaling drives mesenchymal stem cell and fibroblast differentiation into myofibroblasts that secrete angiogenesis, proliferation and motility promoting growth factors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor β (TGF-β) [253-256]. The complex interactions between cancer cells and leukocytes are often called of “Cancer Immunoediting”, and refers to the fact that although the immune system acts as an extrinsic tumor suppressor, it may also be hijacked by the tumor in order to promote its development, invasion and metastasis [257, 258]. The process is characterized by an elimination, equilibrium and escape phases [259], often referred to as the seventh hallmark of cancer [260, 261]. So instead of protecting against carcinogenesis, leukocytedriven chronic inflammation promotes tumor proliferation, progression and invasion, leading to the release of factors that not only promote the accumulation of genetic Chapter 1. General Introduction – Breast Cancer 21 mutations in malignant cells as it also recruits other immune cells that might further be subverted by the tumor [259, 262, 263]. Tumor cells also secrete CSF-1 that attract macrophages to the stroma, than by its turn produce multiple angiogenesis and proliferation factors and pave the stroma for eventual cancer cell metastasis [264-267]. High macrophage infiltration, particularly in areas of necrosis and increased vascular density, is associated with a worse disease-free survival prognosis [265, 268, 269]. Macrophage requirements for both tumor progression and metastasis have been demonstrated in a transgenic mouse model susceptible to mammary cancer with a null mutation in the CSF-1 gene with delayed invasive and metastatic carcinoma. Transgenic expression of CSF-1 restored the invasion and metastasis process, and was associated with an increased infiltration of macrophages in the primary tumor [270]. Macrophage activity is also controlled by other immune system cells, with helper T-cells, through interleukin (IL)-4 secretion, playing a particularly preponderant role in macrophage polarization towards the tumor-associated macrophage (TAM) phenotype [271-274]. In turn, monocytic myeloid derived suppressor cells (MDSCs), through an elevated expression of arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS), non-specifically halt T-cell function and proliferation while recruiting regulatory T-cells (Tregs) to the tumor microenvironment [275, 276]. Macrophages are thought to facilitate tumor progression by two mechanisms:  Macrophages promote the migration of breast epithelial cells towards blood vessels by a macrophage-tumor cell feedback loop. Malignant breast epithelial cells produce CSF-1 that attract colony stimulating factor 1 receptor (CSFR)- positive macrophages, than in its turn secrete EGF, promoting the proliferation of EGFR-positive malignant cells [267, 277, 278]. IL-6 also participates in breast cancer cell-MDSC paracrine signaling. IL-6 expression by malignant breast epithelial cells recruits MDSCs to the primary tumor and preferential metastatic niches. Conversely, these MDSCs also secrete IL-6 and IL-6 receptor (R) subunit α, promoting a positive feedback loop of increased tumor microenvironment IL-6 [279].  Perivascular macrophages in the invasive front are also in close proximity to tumor vessels, and this type of tissue arrangement predicts distant metastasis independently of lymph node status [280]. Recently, a study has suggested that this process may be triggered by direct physical contact between macrophages and tumor cells with consequent formation of RhoA-dependent invadopodia and migration [281]. Chapter 1. General Introduction – Breast Cancer 22 Macrophages probably constitute the tumor microenvironment cell type most associated with tumor progression [268, 282, 283]. Mahmoud et al. demonstrated that high numbers of cluster of differentiation (CD)68-positive macrophages is associated with worst breast cancer-specific survival [282]. On the other hand, overexpression of the macrophage migration inhibitory factor (MIF) was associated with an overall better prognosis [284]. Tregs further contribute to the tumor microenvironment immunosuppression. They are selected by the production of reactive oxygen species (ROS) and other inflammatory factors that also assist their immunosuppressive activity by disturbing the redox balance of other immune cells [285-288]. In primary tumors and lung metastasis the frequency of glycan-binding protein galectin-1 (Gal1) positive cells was found to be positively correlated with Treg numbers and upregulated by transforming growth factor-β (TGF-β). Stromal and tumor cell secreted Gal1 binds to the surface glycoproteins of other immune cells and shifts them towards an immunosuppressive phenotype [289]. Other barriers preventing a functional lymphocyte cancer cell recognition and tumor elimination include the sequestration of tumor antigens and major histocompatibility complex (MHC) molecules and loss of costimulatory factors needed for a proper cytotoxic CD8+ T-cell activity [290292]. A decreased number of CD4+ Th1 T-cells or their precursors, resulting in a skewing of the normal Th1/Th2 ratio and decreased cooperation with cytotoxic CD8+ T-cells may also contribute to the failure to suppress tumor growth [293, 294]. B-cell presence is also frequent in breast tumors as part of the humoral immune response. Recent studies have shown that increased number of B-cells represents an independent indicator for survival [295], particularly in triple-negative (TN) breast cancer patients [240]. Additionally, Levy and coworkers also demonstrated that the number of natural killer (NK) cells may predict recurrence in patients with early stage breast cancer [296]. The invasion of epithelial cells by basement membrane breach and the accumulation of leukocytes and cancer-associated fibroblasts (CAFs) results in a dramatic remodeling of the ECM. The ECM functions as an intermediate in signaling communication between the several cell types, and in turn, these cells can regulate both its composition and structure [297-299]. ECM remodeling through synthesis, degradation, alignment and cross-linking of the matrix [6, 297, 300, 301] affects signaling, potentially leading to tumor cell proliferation and migration, angiogenesis and inflammation [222, 302-304]. Collagen I alignment and stiffening in the tumor microenvironment not only promotes signaling communication between cells as it also sets trails that aid epithelial and stromal cells migration [305-308], and represents an independent negative prognostic Chapter 1. General Introduction – Breast Cancer 23 factor for disease-free survival [309].This ECM rearrangement is promoted by crosslinking enzymes, such as lysyl oxidase, whose expression is modulated by hypoxia inducible factor 1-α (HIF1-α) suggesting an alternative route by which hypoxia within breast tumors may promote metastasis [310-312]. Besides its function as a physical scaffold, collagen I also has the ability to modulate numerous signaling pathways [313, 314], for example, by clustering integrins or regulating SNAIL1 stability to enhance extracellular signal-regulated kinase (ERK) activation [315, 316]. CAFs form an important cellular component of the tumor microenvironment, arising from local fibroblasts and their bone marrow precursors through microenvironment production of TGF-β and platelet-derived growth factor (PDGF) [317-319]. Besides collagen I, CAFs also secrete muscle actin fibers and proteoglycan that aid in the tumor microenvironment reorganization in order to promote tumor outgrowth, growth factor storage, and disruption of the healthy surrounding tissue [320, 321]. Hypoxia is also directly related to tumor microenvironment rearrangement through CAFs: CAFs secrete VEGF and FGF2 as a response to cell membrane mediated Notch receptor-ligand signaling in the presence of HIF1-α [322, 323]. New blood vessels represent not only a source of oxygen and nutrients but also a path for the recruitment of more CAFs, following C-X-C chemokine ligand 12 (CXCL12) secretion into the blood stream. 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General Introduction – Iron Homeostasis in Breast Cancer 55 Chapter 1. General Introduction – Iron Homeostasis in Breast Cancer 56 Chapter 1. General Introduction – Iron Regulation and the Immune System 57 1.3. Iron Regulation and the Immune System Iron is a critical element in life, as a fundamental component of several proteins involved in cell cycle regulation and energy production [1, 2]. Iron homeostasis is tightly regulated at the systemic and cellular levels to avoid free iron-associated toxicity [3, 4]. Current evidence suggests that cells of the immune system participate in the systemic and local regulation of iron homeostasis. This is particularly important given the fact that most pathogenic agents are iron-dependent and the host must modulate iron availability in order to limit its use by microorganisms while assuring the body’s vital iron needs [5-7]. On the other hand, there are various ways by which iron levels can fine-tune the immune system, evidence supported by the observation that several proteins involved in the regulation of iron homeostasis also display immunological properties [8-17]. The average human male adult contains, approximately, 4 g of iron, of which, more or less, 2.5 g is incorporated in the hemoglobin of erythrocytes. By recycling the iron from senescent erythrocytes and delivering it to erythrocyte precursors for hemoglobin synthesis macrophages are recognized as the most relevant leukocytes in iron homeostasis. They are the main players in iron exchanges to the plasma, by favoring or limiting iron export in response to erythropoietic needs or infection/ inflammation [18, 19]. The aging alterations in erythrocytes that may lead to macrophage recognition comprise Band 3 alterations (which is the most abundant erythrocyte membrane protein) [20, 21], phosphatidylserine exposure in the outer cell membrane [22] and increased membrane rigidity [23]. Following red blood cells phagocytosis, heme is catabolized inside macrophages through the action of heme-oxygenase (HO) – 1, releasing iron into the phagosomal lumen that may subsequently be transported to the cytosol by the divalent metal transporter (DMT) natural resistance-associated macrophage protein (Nramp) 1 [2426] and then exported through ferroportin (FPN) 1 (Figure 6) [18, 27]. Alternatively, when in excess, heme may be directly exported via feline leukemia virus subgroup C receptor (FLVCR) into the circulation [28]. Chapter 1. General Introduction – Iron Regulation and the Immune System 64 [43] U.E. Schaible, S.H. Kaufmann, Iron and microbial infection, Nat. Rev. Microbiol., 2 (2004) 946953. [44] M. 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De Sousa, Comparative study between Hfe-/- and beta2m-/- mice: progression with age of iron status and liver pathology, Int. J. Exp. Pathol., 87 (2006) 317-324. [58] J.M. Jones, M. Gellert, The taming of a transposon: V(D)J recombination and the immune system, Immunol. Rev., 200 (2004) 233-248. [59] C.J. Miranda, H. Makui, N.C. Andrews, M.M. Santos, Contributions of beta2-microglobulindependent molecules and lymphocytes to iron regulation: insights from HfeRag1(-/-) and beta2mRag1(-/-) double knock-out mice, Blood, 103 (2004) 2847-2849. [60] A.E. Ten Elshof, G.M. Brittenham, K.A. Chorney, M.J. Page, G. Gerhard, E.E. Cable, M.J. Chorney, Gamma delta intraepithelial lymphocytes drive tumor necrosis factor-alpha responsiveness to intestinal iron challenge: relevance to hemochromatosis, Immunol. Rev., 167 (1999) 223-232. Chapter 1. General Introduction – Iron Regulation and the Immune System 65 [61] E.M. Cardoso, K. Hagen, M. de Sousa, R. Hultcrantz, Hepatic damage in C282Y homozygotes relates to low numbers of CD8+ cells in the liver lobuli, Eur. J. Clin. Invest., 31 (2001) 45-53. [62] E. Cruz, G. Melo, R. Lacerda, S. Almeida, G. Porto, The CD8+ T-lymphocyte profile as a modifier of iron overload in HFE hemochromatosis: an update of clinical and immunological data from 70 C282Y homozygous subjects, Blood Cells Mol. Dis., 37 (2006) 33-39. [63] F.A. Arosa, L. Oliveira, G. Porto, B.M. da Silva, W. Kruijer, J. Veltman, M. de Sousa, Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28T cells, Clin. Exp. Immunol., 107 (1997) 548-554. [64] A. Djeha, J.H. Brock, Uptake and intracellular handling of iron from transferrin and iron chelates by mitogen stimulated mouse lymphocytes, Biochim. Biophys. Acta, 1133 (1992) 147-152. [65] R.M. Ned, W. Swat, N.C. Andrews, Transferrin receptor 1 is differentially required in lymphocyte development, Blood, 102 (2003) 3711-3718. [66] J.D. Kemp, J.A. Thorson, F. Gomez, K.M. Smith, J.S. Cowdery, Z.K. Ballas, Inhibition of lymphocyte activation with anti-transferrin receptor Mabs: a comparison of three reagents and further studies of their range of effects and mechanism of action, Cell. Immunol., 122 (1989) 218230. [67] L.M. Neckers, G. Yenokida, S.P. James, The role of the transferrin receptor in human B lymphocyte activation, J. Immunol., 133 (1984) 2437-2441. [68] L.G. Macdougall, R. Anderson, G.M. McNab, J. Katz, The immune response in iron-deficient children: Impaired cellular defense mechanisms with altered humoral components, J. Pediatr., 86 (1975) 833-843. [69] S.R. Kuvibidila, K.M. Nauss, S.B. Baliga, R.M. Suskind, Impairment of blastogenic response of splenic lymphocytes from iron-deficient mice. In vitro repletion by hemin, transferrin, and ferric chloride, Am. J. Clin. Nutr., 37 (1983) 557-565. [70] J. Jason, L.K. Archibald, O.C. Nwanyanwu, M. Bell, R.J. Jensen, E. Gunter, I. Buchanan, J. Larned, P.N. Kazembe, H. Dobbie, W.R. Jarvis, The effects of iron deficiency on lymphocyte cytokine production and activation: preservation of hepatic iron but not at all cost, Clin. Exp. Immunol., 126 (2001) 466-473. [71] J. Arezes, M. Costa, I. Vieira, V. Dias, X.L. Kong, R. Fernandes, M. Vos, A. Carlsson, Y. Rikers, G. Porto, M. Rangel, R.C. Hider, J.P. Pinto, Non-transferrin-bound iron (NTBI) uptake by T lymphocytes: evidence for the selective acquisition of oligomeric ferric citrate species, PLoS One, 8 (2013) e79870. [72] J.P. Pinto, J. Arezes, V. Dias, S. Oliveira, I. Vieira, M. Costa, M. Vos, A. Carlsson, Y. Rikers, M. Rangel, G. Porto, Physiological implications of NTBI uptake by T lymphocytes, Front. Pharmacol., 5 (2014) 24. Chapter 2 Rationale and Aims Chapter 2. Rationale and Aims 69 Despite recent advances in the knowledge of basic biology, diagnosis and treatment, breast cancer remains the most common type of cancer in women worldwide, with many becoming chemo-resistant, which demands new strategies for disease control. In the last decade we have witnessed a growing body of evidence linking the imbalance of iron homeostasis with the development, behavior and progression of neoplastic diseases. Particularly, in breast cancer, current data suggests that an iron regulatory gene signature associated with minimized epithelial intracellular iron content may predict a favorable outcome, particularly in ER+ patients treated with tamoxifen monotherapy. In spite of the established impact of genetic and epigenetic changes in breast epithelial cells in breast cancer progression, it is now well accepted that these are not sufficient for the acquisition of a fully malignant phenotype. In this respect, gaining insight about the mechanisms by which the cells of the microenvironment promote tumorigenesis is of vital importance. The potential role of cells that take part in systemic iron regulation, such as lymphocytes and macrophages, has not been established in the context of local iron homeostasis in the breast. The main goal of this doctoral thesis was to characterize the iron-associated phenotype of breast epithelial cells, lymphocytes and macrophages in different stages of breast cancer progression. For that purpose, and making use of a series of breast aesthetic reduction specimens, DCIS and IDC, the following studies were performed to assess specific objectives: I. Analyze the iron-related phenotype of epithelial cells, lymphocytes and macrophages in human normal breast and carcinoma samples. Using antibodies against Hepcidin, FPN1, TFR1 and Ferritin (FT) the purpose was to characterize the iron-utilization or iron-donor phenotypes of epithelial cells, lymphocytes and macrophages from primary breast cancer samples, and metastized and non-metastized lymph nodes. The potential association between the expression of these iron-related proteins and classical clinicopathological markers of breast cancer behavior and progression was also a specific aim of this study. II. Evaluate the role of the chemokine CCL2 as a potential modulator of tissue iron status in breast cancer. We aimed at testing if the expression of CCL2 in breast epithelial cells and macrophages could be a modulator of tissue iron deposition and of the ironexporter phenotype observed in lymphocytes and macrophages. Moreover, the Chapter 2. Rationale and Aims 70 association between CCL2 and the clinicopathological markers of breast cancer behavior and progression was also assessed. III. Test the influence of the HFE variants, p.C282Y and p.H63D, on the expression of iron-related proteins. The previously established expression of the iron-related proteins was further evaluated in relation to the presence of HFE variants, in order to test the hypothesis that these are modulators of the expression of iron-related proteins in breast tissue. IV. Analyze the iron-related phenotype of epithelial cells, lymphocytes and macrophages in benign lesions and mammary gland tumors from cats and dogs. Using the same approach as for objective I. we aimed at verifying if the cat and dog are good animal models for human breast carcinogenesis, i.e., if they recapitulate or not the variations observed in the expression of iron-related proteins in human breast cancer. Chapter 3 Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 73 Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 80 Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 81 Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 82 Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 83 Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 84 Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 85 Chapter 3. Local Iron Homeostasis in the Breast Ductal Carcinoma Microenvironment 86 Chapter 4 CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 89 CCL2 EXPRESSION IN BREAST DUCTAL CARCINOMAS: A NOVEL MODULATOR OF LOCAL IRON HOMEOSTASIS? Ana Margarida Rosa1,2,3, Oriana Marques1,2,3,4, Luciana Leite1, Arnaud da Cruz Paula1,5, Alexandra Rêma1, Paula Faustino6, Berta Martins da Silva1,4, Carlos Lopes1,5, Graça Porto 1,2,3,7 1 - Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS), Porto, Portugal; 2 - Basic and Clinical Research on Iron Biology, Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal; 3 - Institute for Research and Innovation in Health (i3S), University of Porto, Porto, Portugal; 4 - Unit for Multidisciplinary Biomedical Research (UMIB), Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal; 5 - Department of Pathology, Portuguese Oncology Institute (IPO), Porto, Portugal 6 - Human Genetics Department, National Health Institute Dr. Ricardo Jorge, Lisboa, Portugal 7 - Hematology Service, Hospital de Santo António - Centro Hospitalar do Porto (HSA-CHP), Porto, Portugal; Corresponding Author Information Ana Rosa, MSc Department of Pathology and Molecular Immunology Institute of Biomedical Sciences Abel Salazar Rua Jorge Viterbo Ferreira, nº 228, 4050-313, Porto, Portugal [email protected] +351 912 294 334 Keywords: Breast; iron; macrophages; CCL2; tumor microenvironment; ferroportin 1 Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 96 Figure 1. Evidence of hemosiderin deposition in a DCIS lesion (a) and in an IDC lesion (b). DABenhanced Perls’ staining of breast tumors, particularly in epithelial (a) and stromal inflammatory cells (b). Original magnification of 200X (a and b) and 400X (inset). Tissue iron deposition was evaluated by assessing the percentage of cases diagnosed within the same lesion presenting iron deposits in epithelial or stromal inflammatory cells. Regarding pure lesions, the presence of hemosiderin deposits in epithelial and stromal inflammatory cells was more evident in carcinomas than in normal tissue (Figure 2). Moreover, statistically significant differences were found for iron deposition in stromal inflammatory cells, between normal and pure DCIS (p=0.011) and Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 97 normal and pure IDC (p=0.001) (Figure 2). The presence of hemosiderin deposits in epithelial and in stromal inflammatory cells was more evident in non-neoplastic tissue adjacent to carcinomas than in normal tissue from reduction mammoplasty samples (p=0.037) (Figure 3). Differences in the percentage of samples presenting iron deposition in stromal inflammatory cells were statistically significant when comparing normal tissue to normal tissue adjacent to IDC lesions (p=0.011) (Figure 3). Figure 2. Presence of hemosiderin deposition in pure lesions of the breast, in epithelial (grey) and in stromal inflammatory cells (black). Chi-square test for iron deposition in stromal inflammatory cells in pure lesions: between normal and pure DCIS p=0.011; between normal and pure IDC p=0.001 (*p< 0.05, **p< 0.01, ***p< 0.001). Abbreviations: EC, epithelial cells; SIC, stromal inflammatory cells; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma. Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 98 Figure 3. Presence of hemosiderin deposition in the tissue adjacent to breast lesions, in epithelial (grey) and in stromal inflammatory cells (black). Chi-square test for iron deposition in stromal inflammatory cells: p=0.011 (*p< 0.05, **p< 0.01, ***p< 0.001). Abbreviations: EC, epithelial cells; SIC, stromal inflammatory cells; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma. Association of CCL2 expression in epithelial and stromal inflammatory cells with malignancy CCL2 has been implicated in breast cancer progression, mainly because of its role as a leukocyte chemoattractant. Comparing with normal tissue, CCL2 immunostaining was higher in tumor samples. Expression of CCL2 in epithelial cells was positively correlated with malignancy (n=80; p=0.007; r= 0.299), as assessed by the Spearman’s rank correlation test. Kruskal-Wallis test was performed and revealed differences in the epithelial CCL2 expression between sub-groups of pure lesions (p=0.022). DunnBonferroni correction showed that statistical significance was only reached when comparing normal to pure IDC lesions (p=0.017) (Figure 4a). Increased expression of CCL2 was already evident in the non-neoplastic tissue adjacent to carcinomas, but we found no statistically significant differences in epithelial CCL2 expression when comparing normal tissue from reduction mammoplasties to non-neoplastic tissue adjacent to carcinomas (p=0.067)) (Figure 4b). Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 99 Figure 4. Median epithelial CCL2 expression in (a) pure lesions of the breast and (b) in non-neoplastic tissue adjacent to breast lesions. (a) Dunn-Bonferroni test: between normal and pure IDC lesions p=0.007. Error bars: 95% CI. (*p< 0.05, **p< 0.01, ***p< 0.001). Abbreviations: DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma. Local infiltration of CCL2-positive macrophages was also analyzed by immunohistochemistry. Total macrophage count presenting CCL2 immunostaining was strikingly higher in pure DCIS lesions and was also positively associated with increased malignancy (n=74; p=0.004; r= 0.335) (Figure 5a). Kruskal-Wallis test revealed statistically significant differences between sub-groups of pure lesions (p<0.001). The DunnBonferroni test revealed that differences in normal samples comparing with pure DCIS and normal comparing with pure IDC samples were statistically significant (Figure 5a). Kruskal-Wallis test revealed no statistical differences in the infiltration of CCL2-positive macrophages between normal tissue from aesthetic reduction mammoplasties and nonneoplastic tissue adjacent to carcinomas (p=0.069) (Figure 5b). Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 100 Figure 5. Infiltration of CCL2-positive macrophages in (a) pure lesions and (b) in non-neoplastic tissue adjacent to breast lesions. (a) Dunn-Bonferroni test: between normal and pure DCIS p<0.001; between normal and pure IDC p=0.001. Error bars: 95% CI. (*p< 0.05, **p< 0.01, ***p< 0.001, versus precedent group) Abbreviations: DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma. Association of total macrophage infiltration with malignancy CD68 immunohistochemistry was performed to facilitate macrophage counting and assess total macrophage infiltration. Macrophage infiltration was more evident in carcinomas than in normal mastectomy samples. According to the Spearman’s test, the number of macrophages was positively associated with malignancy (n=75; p<0.001; r=0.630). Kruskal-Wallis test revealed statistically significant differences between subgroups of pure lesions (p<0.001). Dunn-Bonferroni test revealed significant differences when comparing normal to pure DCIS (p=0.003) and normal comparing to pure IDC (p<0.001). In the hypothetically normal tissue adjacent to carcinomas, the Kruskal-Wallis test revealed no statistically significant differences between sub-groups of lesions (p=0.099). Association of CCL2 expression with macrophage and lymphocyte infiltration Epithelial CCL2 expression was not correlated with total macrophage infiltration (n=75; p=0.609), but was associated with infiltration of CCL2+ macrophages (n=73; p=0.022). Infiltration of CCL2+ macrophages was positively associated with total macrophage infiltration (n=73; p<0.001; r=0.488). On the other hand, expression of this chemokine was positively correlated with the infiltration of CD4+ lymphocytes into the tumor microenvironment (n=67; p=0.019; r=0.286) and with total count of lymphocytes Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 101 (n=67; p=0.025; r=0.273). Infiltration of cytotoxic T-lymphocytes (CD8) or regulatory T cells (CD4/FoxP3) was not correlated with CCL2 epithelial expression or with the infiltration of CCL2+ macrophages. Expression of FPN1 in stromal inflammatory cells We next analyzed FPN1 expression, by immunohistochemistry, in stromal inflammatory cells. Regarding macrophages, considering pure lesions, FPN1 was higher in pure DCIS lesions. Kruskal-Wallis test was performed and statistically significant differences were found between sub-groups of pure lesions (p<0.001). According to the Dunn-Bonferroni correction, differences in FPN1 expression in macrophages were statistically significant when comparing normal samples to pure DCIS (p=0.001) and pure DCIS to pure IDC (p<0.001). When considering the non-neoplastic tissue adjacent to carcinomas, differences in FPN1 expression in macrophages were statistically significant (p=0.009). According to the Dunn-Bonferroni test, differences between sub-groups of lesions were only statistically significant when comparing normal tissue to non-neoplastic tissue adjacent to DCIS lesions (p=0.007). In lymphocytes, according to the Kruskal-Wallis test differences between sub-groups of pure lesions were statistically significant (p=0.011). Dunn-Bonferroni correction revealed differences statistically significant when comparing normal samples to pure IDC (p=0.016). In addition, no statistically significant differences were found between normal tissue from reduction mammoplasties and nonneoplastic tissue adjacent to carcinomas, regarding FPN1 expression in lymphocytes (p=0.079). Association of CCL2 expression and tissue iron deposition We next determined whether CCL2 expression was associated with tissue iron status and explored this association. Samples presenting iron deposition in epithelial cells showed similar median CCL2 levels to samples without iron deposits. On the other hand, epithelial CCL2 expression was associated with iron deposition in stromal inflammatory cells (n=69; p=0.001). In the presence of hemosiderin deposits in lymphocytes and macrophages, median CCL2 expression in epithelial cells was superior, comparing to samples without iron deposition in stromal inflammatory cells. Association of CCL2 and FPN1 expression The remarkable increase in FPN1 expression in stromal inflammatory cells, in carcinomas, and the association obtained between CCL2 expression and iron status, led us to analyze whether CCL2 and FPN1 expression could be correlated. Epithelial CCL2 expression was correlated with FPN1 expression in lymphocytes (n=55; p=0.001; r=0.428), but not in macrophages. Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 102 Association of FPN1 expression with tissue iron status Next, we analyzed whether the increased FPN1 expression observed previously was due to increased iron levels in stromal inflammatory cells. In macrophages and lymphocytes, FPN1 expression was not correlated with iron deposition in stromal inflammatory cells. Influence of HFE variants in CCL2 expression Considering that polymorphisms in the HFE gene are relatively common in the general population and given the fact that they were described as modifiers of CCL2 expression, we next analyzed whether the presence of p.C282Y and p.H63D variants could influence the expression of this chemokine. Of the 83 samples studied, genotyping of the p.C282Y variant was possible for 76 individuals, of which 9 were heterozygous and the remaining homozygous wild type. Genotyping for the p.H63D variant was possible for 69 individuals, of which 36 were homozygous wild type, 27 were heterozygous and 2 were homozygous for the variant. In this study, given the low frequency of individuals with the p.C282Y variant, further statistical tests were not performed. CCL2 expression in epithelial cells was not associated with the HFE H63D variant. Correlation of CCL2 expression with clinico-pathological parameters We next determined whether epithelial CCL2 expression was associated with clinical-pathological parameters of breast cancer behavior and prognosis. CCL2 expression was significantly higher in epithelial cells of ER-negative DCIS cases (n=25; p=0.011). In invasive ductal carcinoma samples, CCL2 expression in epithelial cells was not associated with the estrogen receptor status. Statistical analysis did not show significant associations between epithelial CCL2 expression and classical prognostic parameters, such as tumor size (n=35; p=0.249), molecular subtype (n=55; p=0.215) and lymph node involvement (n=35; p=0.817). Discussion With the present study, we shed new light into the role of stromal inflammatory cells and CCL2, one of the chemokines responsible for their attraction into the tumor microenvironment. A large body of evidence points to the fact that cells in the tumor microenvironment also undergo alterations in response to stimuli sent by epithelial cells, consequently contributing to tumor progression (29). Furthermore, data from different fields of research suggest that in current cancer biology, it should be virtually impossible to address the importance of the tumor milieu without considering the nutritional role of Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 103 iron (30). Recent work performed by our group demonstrated that tumor infiltrating macrophages and lymphocytes display an “iron-donor” phenotype, which suggests that they may act as an iron reservoir, hypothetically contributing to tumor nutrition (18). Results herein described corroborate the hypothesis that stromal inflammatory cells may play a fundamental role in tumor progression, demonstrated by the increased expression of FPN1 in these cells independently of iron levels. In this study, a higher percentage of breast cancer samples presented hemosiderin deposits in epithelial tumor cells, in comparison to normal samples. Due to their incessant proliferation, neoplastic cells have high nutritional requirements, which results in homeostatic deregulation favoring higher intracellular concentrations of iron (9). As first described by Pinnix and colleagues, the deregulation of the ferroportin/hepcidin axis may be central in breast tumor progression (8). Moreover, Chen and co-workers, validating a previous work by Zhang and co-workers (11), reported that FPN1 reduction in epithelial cells was associated with increased intracellular iron levels, which suggests that actively proliferating neoplastic cells have a constant supply of iron, necessary for metabolic reactions (31). Remarkably, we detected increased iron deposition not only in the established malignant lesions, but also in the hypothetically normal tissue adjacent to the representative breast lesion. These results suggest that the non-neoplastic tissue adjacent to carcinomas already present alterations that predispose the microenvironment to acquire more iron for metabolic processes. So, iron deregulation may occur previously to the establishment of lesions and is already present in non-malignant lesions, such as hyperplasias, therefore facilitating epithelial cell proliferation, dysplasia and the potential accumulation of mutations (1). Our results also demonstrate that increased iron deposition is not limited to epithelial cells and that lymphocytes and/or macrophages, especially in carcinoma samples, also present iron accumulation. Lymphocytes are capable of uptaking nontransferrin-bound iron, as demonstrated in a recent study by Pinto and colleagues (32). Macrophages are also specialized cells in handling iron (16) and the regulation of iron homeostasis performed by these cells is possibly not restricted to the systemic level, but also occurs locally. In response to the presence of heme, as a consequence of erythrophagocytosis, FPN1 expression in macrophages is upregulated, independently of hepcidin (33). Based on several evidences, de Sousa hypothesized that in situations of increased blood flow and in the presence of heme, namely in angiogenesis, macrophages mimic this process, by delivering iron to facilitate tumor growth (30). As suggested by de Sousa (30) and supported by the studies previously presented, circulating immune cells may have a surveillance role in controlling potential iron toxicity (34). Tumor-associated macrophages, characterized as M2-like macrophages, act as iron-deficient and are Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 104 characterized by higher FPN1 expression. Recalcati and colleagues reported that M2 macrophages are capable of exporting iron in vitro, which may exacerbate the neoplastic disease by supplying iron to actively proliferating tumor cells (35). As also reported in the work here presented, increased FPN1 expression in lymphocytes and macrophages, particularly in the “normal” to ductal carcinoma in situ transition, may reinforce the iron exporting phenotype suggested for stromal inflammatory cells. Chemokines are best known for their ability to induce cell migration and significantly contribute to cancer progression and metastasis (36). Taking this into consideration, expression of CCL2 in epithelial and stromal inflammatory cells was analyzed by immunohistochemistry. In this study, an increased expression of CCL2 in epithelial and stromal inflammatory cells was observed with increasing malignancy. Mantovani was the first to report that tumor-derived chemokines could be responsible for the attraction of monocytes into the tumor nest, where they could enhance tumor progression, by supplying angiogenic factors and promoting growth (37). The evidence of a significant association between the expression of epithelial CCL2 and the infiltration of CCL2-positive macrophages consolidates the idea of a paracrine signaling pathway. Several authors demonstrated the existence of this pathway, in which tumor cells produce CCL2, responsible for the egress of CCR2-positive monocytes from the bone marrow into the tumor area (38). Tissue macrophages also secrete CCL2, recruiting more macrophages, as demonstrated by Fujimoto and colleagues (39). Our study demonstrates that the infiltration of CCL2-positive macrophages was positively associated with total macrophage infiltration. These results suggest that, as proposed by Fujimoto and colleagues, CCL2 secreted by macrophages, attracted by tumor secreted CCL2, is accountable for the attraction of macrophages from the bone marrow into the breast tumor milieu (39). Epithelial expression of CCL2 was also positively correlated with the infiltration of CD4+ lymphocytes and total lymphocyte infiltration. Although CCL2 is considered to be a monocyte chemoattractant, some studies report that this chemokine may also be an agonist for lymphocytes. Studies performed by Owen and colleagues reported colocalization of the CCR2, the CCL2 receptor, and the pan T cell marker CD3 (40). Moreover, results from a study using a melanoma cell line incubated into mice, suggest that the migration of lymphocytes is dependent on CCL2 produced by tumor cells (41). The influence of iron status in CCL2 expression was already demonstrated by Mitchell and colleagues (21) and in other studies not related to neoplastic disease (22-25). In our study, the presence of iron deposits in stromal inflammatory cells was positively Chapter 4. CCL2 Expression in Breast Ductal Carcinomas: a Novel Modulator of Local Iron Homeostasis? 105 associated with increased epithelial CCL2 expression. This significant association suggests iron as a putative driving force to enhance CCL2 expression in the breast tumor environment. FPN1 expression can be regulated at the transcriptional (by iron deficiency hypoxia, heme and inflammatory cytokines), post-transcriptional (by the IRP-IRE system) or post-translational levels (by hepcidin) (42). Delaby and colleagues demonstrated that FPN1 expression in macrophages and lymphocytes was mainly regulated by iron levels, putatively by the IRP-IRE system (33). In our study FPN1 post-transcription is not regulated by iron levels, suggesting the existence of an alternative signal in the regulation of the iron exporter in breast cancer. Furthermore, expression of FPN1 in lymphocytes, which had been shown to be independent from the iron deposition in stromal inflammatory cells or hepcidin regulation (data not shown), was significantly correlated with CCL2 expression in epithelial cells. These results led us to propose the existence of a mechanism, in which epithelial cells produce CCL2, whose expression may be putatively enhanced by increased iron deposition in stromal inflammatory cells, resulting in the attraction of circulating macrophages and lymphocytes into the tumor site. When these immune cells arrive to the local, FPN1 expression is upregulated, at least partially by the expression of CCL2, and iron is supplied to malignant cells. CCR2-positive macrophages, attracted to the tumor milieu, are responsible for the production of more CCL2, resulting in more infiltrating stromal inflammatory cells (Figure 6). In conclusion, CCL2 may also play a role in regulating tumor iron nutrition and progression, beyond its function as a chemoattractant. Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 113 HFE VARIANTS AND THE EXPRESSION OF IRON-RELATED PROTEINS IN BREAST CANCER INFLAMMATORY STROMAL CELLS Oriana Marques1-4; Ana Rosa2,-4; Luciana Leite2; Paula Faustino5; Alexandra Rêma2; Berta Martins da Silva1,2; Graça Porto2-4,6; Carlos Lopes2,7 1Unit for Multidisciplinary Biomedical Research (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; 2Pathology and Molecular Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; 3Basic and Clinical Research on Iron Biology, Molecular and Cell Biology Institute (IBMC), University of Porto, Porto, Portugal; 4Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Porto, Portugal; 5Human Genetics Department, National Health Institute Dr. Ricardo Jorge, Lisboa, Portugal; 6Hematology Service, Hospital de Santo António, Centro Hospitalar do Porto, Porto, Portugal; 7Department of Pathology, Portuguese Oncology Institute (IPO), Porto, Portugal Corresponding Author Information Reprint Address: Marques, O; E-mail: [email protected] Univ Porto, Lab Immunogenetics & Autoimmu and NeuroScien, Unidade Multidisciplinar Invest Biomed, Inst Ciencias Biomed Abel Salazar Rua Jorge Viterbo Ferreira 228,Edif 2 Piso 4, P-4050313 Oporto, Portugal. Fcomp-01-0124-FEDER-015896 Keywords: breast cancer; HFE; iron Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 114 Abstract The association of HFE major variants with breast cancer risk and behavior has been a matter of discussion for a long time. However, their impact on the expression of iron-related proteins in the breast cancer tissue has never been adressed. In the present study, hepcidin, ferroportin 1, transferrin receptor (TFR1), ferritin expression and tissue iron deposition were evaluated in a sample collection of invasive breast cancers according to the patients’ HFE genotype. Heterozygous patients for the p.C282Y variant presented a higher expression of hepcidin in lymphocytes and macrophages than wild-type or single p.H63D carrier IDC patients. An increased expression of TFR1 in all the cell types analyzed was also observed in p.C282Y/H63D compound heterozygotes, only. A differential impact of the two HFE variants was further noticed with the observation of a significantly higher percentage of p.C282Y heterozygous patients presenting tissue iron deposition in comparison to p.H63D heterozygotes. No significant associations were found in this study between HFE variants and the classical clinicopathological markers of breast cancer behavior and prognosis. Although limited by a low sampling size, our results suggest that HFE major variants could play a role in breast cancer progression not by influencing systemic iron homeostasis but rather by differentially modulating the local cellular expression of iron-related proteins and tissue iron deposition. Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 115 Introduction Iron is an essential trace element for the human body, as a critical component of several signaling, transporter and storage molecules involved in energy production and intermediate metabolism (Andrews, 1999; Ganz and Nemeth, 2011). However, its characteristic chemistry contributes to the formation of hazardous molecules, such as hydroxyl radicals and hydrogen peroxide, when in excess (Fenton, 1894; Halliwell and Gutteridge, 1992; Kalinowski and Richardson, 2005). Although most organisms have the proper mechanisms to regulate iron homeostasis to avoid free iron toxicity, current knowledge suggests that the deregulation of its regulatory mechanisms may contribute to a number of chronic diseases (Kell, 2009). Iron is thought to promote carcinogenesis through iron-induced oxidative stress, modulation of signaling networks associated with malignancy and by providing selective advantage to rapidly growing tumor cells (Omary et al., 1980; Cermak et al., 1993; Eaton and Qian, 2002; Benhar et al., 2002; Kowdley, 2004; Galaris et al., 2008). HFE is a MHC class-I like protein that acts as a gatekeeper of systemic iron homeostasis by controlling hepatic hepcidin levels (Schmidt et al., 2008; Vujic Spasic et al., 2008). Hepcidin, in turn, maintains normal plasma iron levels by regulating iron release from cells through the binding to its receptor, the iron exporter ferroportin 1 (Nemeth et al., 2004; Nemeth and Ganz, 2006). A proposed molecular mechanism positions HFE and Transferrin Receptor 1 (TFR1) in an iron-sensing complex which is disrupted by binding of circulating holotransferrin with a higher affinity for TFR1 (Goswami and Andrews, 2006). Upon TFR1 dissociation, HFE is able to relocate to TFR2 and interact with the bone morphogenetic protein (Bmp) co-receptor Hemojuvelin (Goswami and Andrews, 2006; D'Alessio et al., 2012), involved in signal communication upon binding of the Bmp ligands, and whose interaction leads to the activation of hepcidin transcription (Miyazono et al., 2005; Babitt et al., 2006; Babitt et al., 2007). However, previous evidences from others suggest that HFE may also act a regulator of iron uptake through its direct interaction with the TFR1 (Feder et al., 1996b; Lebron et al., 1998; Waheed et al., 2002). HFE gene variants p.C282Y and p.H63D are very common in normal European derived populations. The p.C282Y variant disrupts the association of HFE with β-2 microglobulin, reducing the cellular surface expression of HFE (Feder et al. , 1996b; Feder et al., 1997; Waheed et al., 1997). This alteration is responsible for the large majority of hereditary hemochromatosis cases (Feder et al. , 1996b). The p.H63D variant is believed to lower the HFE protein affinity for TFR1 (Gray et al., 2009), but its association with iron overload is controversial (Aguilar-Martinez et al., 2001; Kelley et al., 2014; Porto et al., 2015). Although epidemiological studies have been inconsistent in supporting an association between HFE Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 116 major variants and an increased risk for breast cancer (Nelson et al., 1995; Beckman et al., 1999; Kallianpur et al., 2004; Abraham et al., 2005; Osborne et al., 2010), it is plausible to assume that, by interfering with the cellular and tissue iron homeostasis, they may affect the cancer cell phenotype. We have previously shown that the deregulation of iron-related proteins in breast cancer, more specifically hepcidin, ferroportin 1 (FPN1), TFR1 and ferritin (FT), is not restricted to epithelial cells, but also extends to cells of the tumor microenvironment (Marques et al., 2016). To our knowledge, no other group has attempted to verify if the HFE major variants had an impact on the expression of iron-related proteins in the neoplastic context. Materials and Methods Sample Characterization A previously characterized cohort of human breast tissue samples, archived at the Pathology Service at Centro Hospitalar do Porto, was used in this study. This cohort consisted of 120 samples, including 56 cases of invasive ductal carcinomas (IDC), 14 cases of ductal carcinomas in situ (DCIS) and 49 samples without evidence of breast disease obtained from breast reduction aesthetic surgery. The study has been previously approved by the local Research Ethics Committees, as part of a more extended study (see Marques et al., 2016). Clinicopathological features, such as histological diagnosis, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) statuses were retrieved from interin pathology reports. ER, PR and HER-2 status were assessed by immunohistochemistry. HER-2 ambiguous results were confirmed by FISH. Tissue Microarray Construction and Immunohistochemistry Tissue microarray construction and immunohistochemistry for hepcidin, FPN1, TFR1 and FT for this cohort have been extensively described before (Marques et al., 2016). Immunostaining for hepcidin, FPN1, TFR1 and FT was evaluated in epithelial cells, lymphocytes and macrophages using the same semi-quantitative evaluation method as before (Marques et al., 2016). Cores from the same donor tissue were grouped and their mean score for each variable calculated. Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 117 DAB-enhanced Perls’ Prussian Blue Staining To evaluate the presence of iron deposition in breast samples, DAB-enhanced Perls’ Prussian Blue was performed, adapted to the Van Duijn protocol (Van Duijn et al., 2013). DNA Extraction and HFE Genotyping Genomic DNA was extracted from FFPE breast sections according to the Ultraprep Tissue DNA kit (AHN Biotechnologie, Nordhausen, Germany) recommended procedures. PCR was carried out in 15.5μL reaction volumes, containing 2μL of the genomic DNA template, 7.5μL of MasterMix DNA polymerase, 1μL of Q-solution (Qiagen Multiplex PCR kit, Valencia, CA, USA) and 1μL of each of sense and antisense primers. For the detection of the p.C282Y variant the following primers were used: 5'- CAAGTGCCTCCTTTGGTGAAGGTGACACAT-3' as the forward primer and 5’- CTCAGGCACTCCTCTCAACC-3’ as the reverse primer (Metabion, Steinkirchen, Germany). Following the verification of the 343 bp fragment amplification RsaI was used for restriction. For the HFE p.H63D variant, the following forward and reverse primers’ sequences were used: 5’-ACA TGG TTA AGG CCT GTT GC-3’ and 5’-GCC ACA TCT GGC TTA AAA TT-3’ (Metabion, Steinkirchen, Germany). In turn, these primers amplify a fragment with 294 bp that was then restricted by MboI. Primers for detection of variants in the HFE gene were chosen according to the work of Feder et al. (Feder et al., 1996a). The PCR program included a step of 95ºC, for 15 minutes followed by 36 cycles of denaturation at 94ºC for 30 seconds, annealing at 58ºC for 90 seconds and extension at 72ºC for 90 seconds. Reaction was extended in the final for 10 minutes at 72ºC. Statistical Analysis Data were analyzed with IBM SPSS Statistics Version 18.0 (SPSS Inc., IBM, Chicago, IL, USA). Sample distributions were compared by the Kruskal-Wallis test followed by post-hoc testing whenever the omnibus testing was significant. Pearson’s ChiSquare was used to evaluate the differences between categorical variables. In figures, experimental errors are shown as 95% Confidence Intervals (CI). Statistical significance was accepted at p< 0.05. Once no p.C282Y heterozygotes were found within the aesthetic breast reduction population, comparisons for this variant were restricted to breast cancer cases. Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 118 Results Expression of Iron-Related Proteins No significant differences were found in the expression of the analyzed iron-related proteins between wild-type individuals and p.H63D carriers included in the aesthetic breast reduction population for all cell types. Similarly, no significant differences were found for the expression of these proteins in DCIS among all the genotypes considered. In invasive breast carcinoma (IDC) cases, however, the expression of hepcidin in lymphocytes and macrophages was significantly higher in patients carrying the p.C282Y variant (p< 0.05; Figure 1A) relative to both wild type and p.H63D carriers. No further differences were found regarding the expression of FPN1, TFR1 and FT in IDC cases (Figure 1B-D). Figure 1. Iron-related proteins in invasive breast carcinomas according to HFE genotype. Median hepcidin (A), FPN1 (B), TFR1 (C) and FT (D) expression, in epithelial cells, lymphocytes and macrophages, in relation to the presence or absence (WT) of the p.C282Y and p.H63D variants. Scores ranged from 0 to 15 and errors bars present 95% CI. Abbreviations: WT, Wild-Type; FPN1, ferroportin 1; TFR1, transferrin receptor 1; FT, ferritin; EC, Epithelial Cells; Ly, Lymphocytes; M0, Macrophages Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 119 To further clarify the relative impact of the two variants in the results observed, we focused the analysis on a sub-sample of p.C282Y/p.H63D compound heterozygotes. Remarkably, from the 9 p.C282Y carriers with IDC, 5 were compound heterozygotes. Although these did not differ in general from the other p.C282Y/WT IDC patients in terms of the expression of the iron-related proteins, they differed significantly from non-p.C282Y carriers not only by a higher expression of hepcidin in lymphocytes and macrophages (as described in the whole p.C282Y carrier population) but they showed, in addition, an increased expression of TFR1 in all the cell types analyzed (Table 1). Table 1. Expression of iron-related proteins in HFE p.C282Y/p.H63D compound heterozygous IDC patients is increased in comparison with patients without the p.C282Y variant. * represents significant differences (p< 0.05) for comparison with the non-p.C282Y carriers group. Abbreviations: IDC, invasive ductal carcinoma; SEM, Standard Error of the Mean; EC, Epithelial Cells; Ly, Lymphocytes; M0, Macrophages; NS, Non-Significant Iron-Related Proteins (Mean ± SEM) non-p.C282Y carriers (n= 47) p.C282Y/p.H63D heterozygous compounds (n= 5) p.C282/WT heterozygous (n= 4) Hepcidin EC 1.06 ± 0.28 3.4 ± 2.93 6.38 ± 3.33 Ly 0.80 ± 0.16 2.17 ± 0.69* 2.00 ± 0.58 M0 1.56 ± 0.32 3.60 ± 0.98* 3.83 ± 1.30 FPN1 EC 5.54 ± 0.39 6.11 ± 1.02 6.13 ± 1.71 Ly 6.67 ± 0.54 8.43 ± 1.06 4.28 ± 1.88 M0 6.17 ± 0.43 7.90 ± 0.75 4.61 ± 1.69 TFR1 EC 7.25 ± 0.51 11.00 ± 1.72* 7.65 ± 1.98 Ly 3.70 ± 0.32 5.80 ± 0.97* 4.33 ± 0.88 M0 6.14 ± 0.38 9.68 ± 1.42* 7.50 ± 2.25 FT EC 10.09 ± 0.35 8.92 ± 0.74 9.06 ± 0.60 Ly 8.89 ± 0.24 8.50 ± 1.07 6.88 ± 0.59* M0 14.83 ± 0.12 15.00 ± 0.00 14.17 ± 0.83 Chapter 5. HFE and the Expression of Iron-Related Proteins in Breast Cancer 120 Tissue Iron Deposition Regarding tissue iron deposition in invasive breast carcinomas, a significantly lower percentage of p.H63D carrier IDC patients presented iron deposits in epithelial and stromal inflammatory cells, compared with the two other genotypes considered (p< 0.05; Figure 2). Differences between wild-type and p.C282Y heterozygotes were not statistically significant. From the p.C282Y carrier IDC patients displaying iron deposits in epithelial cells, all of them were compound heterozygotes and from the ones with iron deposition in stromal inflammatory cells, 4 out of 5 were compound heterozygotes. Figure 2. Iron deposition in invasive breast carcinomas according to the HFE genotype. Percentage of invasive breast cancer cases presenting iron deposition in epithelial (black) and stromal inflammatory (grey) cells, in relation to the HFE genotype. Abbreviations: WT, Wild-Type Clinicopathological Data Hormone receptor and HER2 status, lymph node involvement and tumor size were available from the interin records and were also analyzed regarding the HFE genotype. None of the different genotypes were associated with any of the considered variables of poor outcome (Table 2). 121 Table 2. Clinicopathological features of breast cancer patients according to their HFE genotype. Abbreviations: DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; ER, estrogen receptor; PR, progesterone receptor, HER2, Human Epidermal growth factor Receptor 2; LN, lymphnode; IQR, interquartile range; NS, non-significant DCIS IDC Factors WT p.H63D carriers p.C282Y carriers p WT p.H63D carriers p.C282Y carriers p n (%) n (%) n (%) n (%) n (%) n (%) ER status, n (%)a NS NS negative 2 (28.6%) 1 (25.0%) 2 (66.7%) 7 (22.6%) 3 (16.7%) 3 (37.5%) positive 5 (71.4%) 3 (75.0%) 1 (33.3%) 24 (77.4%) 15 (83.3%) 5 (62.5%) PR status, n (%)a NS NS negative 3 (42.9%) 2 (50.0%) 2 (66.7%) 8 (25.8%) 5 (27.8%) 4 (50.0%) positive 4 (57.1%) 2 (50.0%) 1 (33.3%) 23 (74.2%) 13 (72.2%) 4 (50.0%) HER2 status, n (%)a NS NS negative 4 (57.1%) 1 (33.3%) 2 (66.7%) 24 (77.7%) 13 (76.5%) 4 (50.00%) positive 3 (42.9%) 2 (66.7%) 1 (33.3%) 7 (22.6%) 4 (23.5%) 4 (50.00%) LN metastasis, n (%)a NS non-metastized 17 (56.7%) 4 (22.2%) 4 (50.0%) metastized 13 (43.3%) 14 (77.8%) 4 (50.0%) Median tumor size (IQR)b 1.00 (1.00 - 2.00) 1.00 (1.00 - 2.00) 1.00 (1.00 - 1.50) NS a Pearson Chi-Square b Kruskal Wallis Test