L’endométriose est une maladie gynécologique pour laquelle l’exposition à certains contaminants chimiques environnementaux est évoquée parmi les facteurs de risque associés. Les conclusions des études épidémiologiques existantes restent toutefois non convergentes. Leur hétérogénéité en termes de lésions décrites, de méthodologie et d’effectifs contribuent à ce constat, de même que l’étendue limitée des marqueurs d’exposition considérés dans ces études. Nous avons réalisé une étude cas-témoins appariés à partir d’une bio-collection de 113 patientes réunissant68 cas de patientes opérées d’endométriose profonde et 45 patientes témoins. Un ensemble unique de 78 polluants organiques persistants a été recherché, incluant dioxines, polychlorobiphényles, retardateurs de flamme polybromés, et pesticides organochlorés. Les niveaux d’exposition interne des sujets ont été mesurés à la fois dans les tissus adipeux pariétal et épiploïque ainsi que dans le sérum. La distribution de ces différents polluants au sein de ces trois compartiments a tout d’abord été caractérisée. Celle-ci a permis la prise en compte encore très rare de l’équilibre entre compartiments de stockage et compartiment circulant, ce rapport de concentration apparaissant comme un potentiel indicateur additionnel permettant d’affiner d’éventuels liens de causalité entre exposition chronique à des dangers chimiques et pathologie chez l’homme. Certains des contaminants ciblés sont ensuite apparus significativement associés à l’endométriose profonde, la stratification plus fine de notre population de cas indiquant un lien d’autant plus significatif en présence d’endométriome. Les mécanismes sous-jacents de cette association restent toutefois à élucider. / Endometriosis is a gynecological disease for whichexposure to some environmental chemicals is evocatedamong the associated risk factors. Epidemiological studies are however globally non convergent and finally fairly conclusive. Their heterogeneity in terms of lesion localization and sub-phenotype, methodology, size and nature of the populations studied, as well as the limited number of monitored markers of exposure contribute to this situation. We realized a matched case-control study based on a biocollection of 113 patients including 68 patients suffering of deep endometriosis and 45 controls. We characterized the internal exposure levels of an extended range of around 78 persistent organic pollutants (including dioxins, polychlorobiphenyls, brominated flame retardants and organochlorine pesticides). Internal level exposures were measured in three biological compartments (omental fat, subcutaneous fat and serum). First, the distribution of these chemicals was characterized within these compartments. These extended exposure data from deep infiltrating endometriosis patients are the first ones available for France and give a new insight about the equilibrium of chemicals between storage and circulating compartments that should be further considered as a potential indicator permitting to establish a possible association between a chronic exposure to chemical hazards and human pathology. Afterwards, some of the targeted chemicals appeared significantly associated with deep endometriosis. A sub-stratification of our case population indicated a more significant relationship with the presence of endometrioma. Underlying mechanisms remain to be determined.

INTRODUCTION: Melanoma is the most deadly skin cancer with mortality dependent on the extent and location of metastases. Lymphatic metastasis occurs early in melanoma, and tumor-associated lymphatic vessel area correlates with melanoma progression. Recently, the discovery of checkpoint inhibitors has drastically changed the treatment strategy and survival rates in melanoma. Neuropilin-2 (NRP2) is a potential common target in melanoma cells, tumor-associated lymphatic endothelial cells (LECs) and tumor-infiltrating lymphocytes (TILs). NRP2 is a cell surface receptor with competing stimulatory ligands (VEGF-A/-C) and inhibitory ligands (SEMA3F/G). AIM: The goal of this study was to investigate the role of NRP2 in both melanoma cells and the melanoma microenvironment (LECs, TILs) and to examine the effect of semaphorin 3F (SEMA3F) on the tumor cells as well as an immune modulator. RESULTS: Mouse and human melanocytes expressed NRP2 but not other vascular endothelial growth factor (VEGF) receptor tyrosine kinases in vitro and in vivo. NRP2 protein expression, as analyzed by immunohistochemistry, was upregulated in human metastatic melanoma sections. Treatment of melanoma cells in vitro with SEMA3F inhibited migration and phosphorylation of protein kinase B (AKT) but did not inhibit cell viability. SEMA3F also increased programmed cell death-ligand 1 (PD-L1) expression in melanoma. Syngeneic B16F10 melanoma did not grow in global NRP2 knockout (KO) mice but did grow in wild-type mice. In addition, mice inoculated with B16F10 were treated with SEMA3F or phosphate-buffered saline (PBS) by mini-osmotic pumps. Resulting tumors were analyzed histologically for microvessel density and presence of TILs (number and subtype). CONCLUSIONS: Expression of NRP2 protein positively correlated with melanoma progression in human patient samples. NRP2 functions differently in melanoma tumor cells than in host stromal cells (endothelial cells [ECs], LECs). In melanoma, NRP2 is not a VEGF receptor but responds to the ligand, SEMA3F. Alternately, NRP2 appears to be an important VEGF-A/-C co-receptor in tumor-associated angiogenesis and lymphangiogenesis, as demonstrated in the NRP2 transgenic mice studies. SEMA3F inhibited tumor cell migration but increased PD-L1 expression. Systemic treatment with purified SEMA3F protein in melanoma preclinical trials inhibited melanoma growth and microvessel density. Taken together, these results suggest that exploiting the NRP2/SEMA3F signaling axis may be a novel treatment strategy to be used in combination with existing immunotherapy in melanoma. / 2020-07-03T00:00:00Z

Chronic inflammation is associated with carcinoma development in several clinical settings, and we sought to investigate the role of T cells in this phenomenon using the DMBA/TPA two-stage chemical carcinogenesis protocol. We demonstrate that, paradoxical to models of immunosurveillance, wild-type (WT) mice have a markedly higher rate of tumor formation relative to strains lacking CD8+ T cells. Adoptive transfers of antibody-coated magnetic bead-enriched peripheral CD8+ T cells into TCRáâ-/- mice confirmed that the increased mean tumor area and progression to carcinoma was attributable to the presence of CD8+ T cells. All analyzed strains of mice in which the CD8 compartment was intact (WT, CD4-/-) showed significant increases in tumor susceptibility. Putative tumor-promoting (T-pro) cells (TCRáâ+CD8+CD44+CD62L- tumor infiltrating lymphocytes, TILs) were directly compared to their phenotypic equivalents in peripheral blood lymphocytes (PBLs). In WT and CD4-deficient mice, CD8+ TILs consistently revealed a markedly higher relative expression, by RT-PCR, of IFNã, TNFá and COX-2, and a striking decrease in expression of perforin. Cytokine-bead analysis (CBA) comparison of CD8+ and CD4+ TIL in tumors from WT mice confirmed the increased expression by the CD8+ TIL of IFNã and TNFá. To our knowledge, this is the first demonstration of increased carcinogenesis attributable to CD8+ TILs, characterized by their high IFNã, TNFá, and COX-2 production and defective perforin production relative to phenotypically equivalent PBLs. These studies may have mechanistic implications for the role of T cells in inflammation-associated carcinogenesis.

Cancer immunotherapy has become a leading treatment modality in metastatic diseases. Although this novel therapy has changed the therapeutic algorithms and patients' outcomes in multiple malignancies, certain proportions of patients still fail to respond to these approaches. In our studies, we aimed to address the main mechanisms of tumor resistance to cancer immunotherapy. We have systematically defined the main challenges in adoptive cell transfer. We have focused on two key mechanisms of the tumor resistance to immunotherapy: poor trafficking of adoptively transferred immune cells into tumors, and the death receptor-induced apoptosis of the tumor-infiltrating immune cells. In our work, we have gone beyond the tumor tissue and searched for the immune cell populations and novel targets that would help to challenge the two mechanisms of resistance. Our data uncovered the therapeutic potential of the paratumoral tissue compartments and, thus, provided new avenues on how to challenge solid tumors by immunotherapy.

Breast cancer (BC) was not initially considered an immunogenic tumor; however, recent data show that immune-related factors are associated with patient prognosis and the response to treatment. Several large adjuvant clinical trials have shown that tumor infiltrating lymphocytes (TIL) are significantly associated with a better prognosis and can also predict responsiveness to pre-operative chemotherapy, particularly in the triple negative (TN) & HER2+ BC subtypes (Carsten Denkert et al. 2010; Loi et al. 2013a). Recently, the presence of ectopic lymph node-like structures characterized by distinct T and B cell zones, called tertiary lymphoid structures (TLS), were identified adjacent to the tumor (Gu-Trantien et al. 2013) in 60% of BC (Buisseret et al. 2017b) and linked with a good prognosis (Gu-Trantien et al. 2013). The mechanisms involved in TLS formation and activities and their impact on tumor immunity is relatively unknown. TIL infiltration and TLS formation are likely regulated, in part, by transcription factors (TF) that control cytokine/chemokine production within the tumor microenvironment (TME) (Pimenta and Barnes, 2014). One such TF, the forkhead box protein 1 (FOXP1) is abnormally expressed in various human tumors and has a known role in regulating immune cell functions. Contradictory data on FOXP1 expression together with a lack of information on its immune regulation led us to explore its role in this tumor type. The first part of this thesis research focused on FOXP1-mediated regulation in BC. Gene/protein analysis was examined in the four BC molecular subtypes, revealing its enriched expression in estrogen receptor positive (ER+) tumors (Luminal A/B). Luminal BC is generally less infiltrated compared with frequently high TIL infiltration in ER negative (ER-) tumors (i.e. HER2+ and TN) [reviewed in (Solinas et al. 2017a) and (Loi et al. 2014)]. We found that high FOXP1 expression in a cohort of untreated primary BC was significantly associated with a lower TIL and fewer TLS compared to FOXP1 low (FOXP1lo) tumors. This observation led us to investigate the effect of FOXP1 on cytokines and chemokines potentially involved in TIL recruitment and/or TLS formation. BC cancer cell lines were used to silence [MCF7; FOXP1hi] or overexpress [MDA-MB-231; FOXP1lo] FOXP1 expression. FOXP1 repression upregulated a number of cytokines and chemokines involved in T and B cell migration and function, while FOXP1 overexpression repressed a majority of the same factors. Expression analysis of the major T and B cell cytokine and chemokine genes was performed for FOXP1lo and FOXP1hi primary BC. These data reveal that FOXP1hi BCs have significant decreases in CXCL9, CXCL10, CXCL11, CXCL13, CX3CL1, CCL20, IL2, IL21, granzyme B and IFNγ and high levels of the immunosuppressive cytokines, IL10 and TGFβ. We next performed a lymphocyte migration assay using primary tumor supernatants prepared from FOXP1lo and FOXP1hi BC finding significantly decreased migration of total CD45+ lymphocytes, B cells, helper (CD4+) and cytotoxic (CD8+) T cells using FOXP1hi compared to FOXP1lo SN. Overall, our data suggest that FOXP1 plays an important role in repressing anti-tumor immune responses by negatively regulating TIL migration directed by specific cytokines and chemokines.The second part of this thesis research focused on the role FOXP1 plays in BC TLS. FOXP1 expression in T and B cell TIL and TLS was evaluated using RT-qPCR, multicolor flow cytometry, immunofluorescence (IF) and immunohistochemistry (IHC) and fresh, fixed and frozen breast tissues. Based on the FOXP1 expression two types of TLS were identified in BC: 1) TLS containing a germinal center (GC-TLS) and 2) TLS lacking a GC (non-GC-TLS). Examination of proteins specifically associated with active humoral immune responses allowed us to identify GC-TLS but not non-GC-TLS as functional. Gene expression analysis of micro-dissected tissues revealed distinct immune profiles that characterize B cell follicles in tonsils and spleen as well as aggregates, non-GC-TLS and GC-TLS in BC. This analysis further demonstrates that ongoing cell-mediated immune responses are associated with GC-TLS. The findings from this thesis research add important information to our understanding of how immune responses are initiated and maintained in BC and provide further insight into the identification and organization of functional immune responses at the tumor site. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Abstract Aptosis denotes a biochemically and morphologically distinct chain of events leading to self-destruction of cell. It is pivotal in the maintenance of tissue homeostasis and also plays a role in neoplasm. In this work, the extent of apoptosis and apoptosis regulating proteins and factors was studied in a total of 94 patients operated for lung carcinoma, including 56 small cell lung carcinomas (SCLC) and 38 large cell lung carcinomas (LCLC). The extent of apoptosis was determined by detecting and counting the relative and absolute numbers of apoptotic cells and bodies using 3'- end labelling of the apoptotic DNA. The extent of apoptosis in SCLC was compared with the cell proliferation activity as determined by Ki-67 immunohistochemistry, with the volume density of necrosis and with the occurrence of immunohistochemically detectable p53 and bcl-2 proteins. In order to test the hypothesis that increased apoptotic activity is connected with neuroendocrine differentiation and with low differentiation degree in LCLC and that it is regulated by bcl-2 family proteins, the extent of apoptosis and tumour necrosis was analysed in relation to the expression of bcl-2 family proteins bcl-2, mcl-1, bax and bak. Apoptosis, tumour infiltrating lymphocytes (TILs), and angiogenesis are important factors that contribute to tumour growth. In the present study immunohistochemical methods were used to investigate the relationships of these factors and their role in the prognosis of the patients with LCLC and SCLC. A remarkably high apoptotic activity was detected in both SCLC and LCLC. The mean apoptotic index in SCLC was 2.70 % and in LCLC 2.49 %. Exceptionally high proliferation activity and high percentage of tumour necrosis was seen in SCLC. 58 % of SCLC showed more than 40 % of Ki-67 positive nuclei, and tumour necrosis was seen in 83 % of the cases. P53 protein accumulation was detected in 38 % and bcl-2 expression in 50 % of SCLC. The extent of apoptosis in SCLC was inversely related to tumour necrosis and p53 protein accumulation. In LCLC, bcl-2 expression was detected in 40 % of the cases. It was associated with neuroendocrine differentiation and predicted favourable prognosis of the patients. A high number of T cells and macrophages with a small number of B cells was detected in both SCLC and LCLC. The occurrence of intratumoural cytotoxic CD8 cells was associated with the occurrence of apoptotic bodies in SCLC. The increased number of intratumoural T cells, CD8-positive cells and macrophages predicted favourable prognosis of the patients with SCLC. In LCLC, an increased number of B cells and macrophages, but not T cells, was associated with better survival. Iaddition to tumour cells, numerous apoptotic bodies could also be found within alveolar macrophages within and close to tumour tissue. In order to test whether such cells could be found in sputum smears and if their presence could be utilised as a marker of malignancy in tumour diagnosis, the occurrence of alveolar macrophages with apoptotic bodies (AMWABs) was analysed in 84 sputum samples and 13 broncho-alveolar lavage (BAL) specimens from patients with and without lung carcinoma. AMWABs could be found in cytological samples of the patients with lung carcinoma. In sputum and BAL specimens, enhanced apoptosis, as measured by an increased number of AMWABs reflected and was indicative of malignancy. This was also true for cytological specimens of the patients even when the actual malignant cells were not found. Therefore the AMWABs served as a marker of pulmonary malignancy.

Introduction: Human papillomavirus (HPV) is changing the prevalence, survival and treatment paradigms in oropharyngeal squamous cell carcinoma (OPSCC). Improved survival of patients with HPV positive compared to HPV negative OPSCC has led to trials of treatment de-escalation. Current HPV detection methods are imprecise, therefore standardised assessment of transcriptionally active HPV in OPSCC is required. Furthermore, the differences in immune characteristics and/or the hypoxia response/effects could explain observed differences in prognosis between HPV positive and negative OPSCC. Rigorous HPV detection and subsequent biomarker evaluation should provide additional information required before introduction of treatment de-escalation in broad patient groupings. Methods: The study cohort was 218 patients with OPSCC who received radiotherapy with curative intent. HPV status was determined on pre-treatment, formalin-fixed paraffin-embedded blocks using: 1) polymerase chain reaction (PCR); 2) in-situ hybridisation (ISH) and 3) immuno-histochemistry (IHC). QuantiGene multiplex assay was designed to detect mRNA of reference sequences of the common high-risk HPV types (16, 18, 33, 35, 45, 52 and 58). HPV detection methods were compared with mRNA quantification. Multimarker IHC of immune cell markers using chromogenic and fluorescent staining was performed, analysed and compared with single marker IHC using automated multispectral image analysis. A validated multiplex IHC method was used for a) chromogenic (CD3, CD4, CD8, and FoxP3) and b) fluorescent (CD8, CD68 and PD1/PD-L1) evaluation in tumour and stroma compartments. Single marker IHC was used to investigate tumour hypoxia markers (HIF-1α and CA-IX) in HPV positive and negative OPSCC. Results: p16 IHC and ISH were the most sensitive and specific, respectively, for classifying HPV status. The combination of the three tests had the highest positive/negative predictive values compared with QuantiGene mRNA detection. Multiplex validation showed that, for serial sections up to 6 μm apart, there were highly significant correlations (P<0.0001) between single and multiplex counts for both chromogenic and fluorescent IHC. Overall there was less variation in cell counts with fluorescent staining when compared to chromogenic staining. Multiplex IHC of TILs in HPV positive and negative OPSCC showed higher infiltration in both tumour and stromal areas of CD3+CD4+ and CD3+CD8+ T cells but not CD4+FoxP3 Tregs in HPV positive compared with HPV negative OPSCC. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). PD-L1 expression was higher in HPV negative OPSCC and this was related to macrophage (CD68) expression of PD-L1. In HPV negative tumours infiltration with CD68+PD-L1 was associated with a good prognosis. HPV negative patients had higher expression of HIF-1α but not CA-IX. High expression of both markers was associated with a poor prognosis irrespective of HPV status. Conclusions: There are other prognostic factors operating in the larger subdivision of HPV positive and negative OPSCC. Precise HPV detection and inclusion of other prognostic factors is required before treatment de-escalation is used. Expression of immune inhibitory factors (PD1/PD-L1) alone without contextualisation with immune cell density is insufficient for patient prognostication and potential selection for therapy using immune checkpoint inhibitors. Hypoxia modification of radiotherapy should be explored in both HPV positive and negative OPSCC.

No description available.

Melanoma is a highly malignant neoplasia with high rates of metastasis in humans and dogs. Regardless of being considered a highly immunogenic neoplasm, the function of the immune system is hampered by the expression of immune checkpoint molecules by the cancer cells. In contrast, soft tissue sarcomas are poorly immunogenic, as Tumor infiltrating Lymphocytes are lacking, or when present they are usually at the periphery of the tumor. Still, soft tissue sarcomas are considered immunosuppressed. Checkpoint molecules from the PD-axis are overexpressed in numerous human malignant neoplasia and have recently gained attention with a few reports in canine tumors. Immunotherapies against these checkpoint molecules have shown great efficacy in humans, but in order to determine translational approaches into canine patients, more research is needed. Here we determined the gene expression of Programed Death receptor-1, and its ligands PD-L1 and PD-L2 in canine tumors with two distinct immune profiles. Our results show that regardless of their immune profiles, melanoma versus soft tissue sarcoma, checkpoint molecules expression was higher in malignant tumors with a higher grade. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal ultrasound focused therapy that induces mechanical stress to the cells, leading to liquefactive necrosis. Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of CD3+ T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasia, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia. / Doctor of Philosophy / Melanoma is a highly malignant tumor and very resistant to therapy for humans and dogs. At the same time, this neoplasia is usually highly infiltrated by cells from the immune system. However, this immune infiltration is often inhibited by molecules expressed by the melanoma cells. In contrast, soft tissue sarcoma is considered poorly immunogenic, as they often contain low levels of immune cell infiltrates but are still considered immune suppressed. In this study, we determined the expression of molecules that inhibit the effect of T lymphocytes, specifically Programed cell death receptor-1, PD-Ligand 1, and PD-Ligand 2 for these neoplasms with distinct immune profiles. We encounter that despite their immune profiles, the expression of these molecules is higher in malignant tumors. Additionally, we evaluated the expression of these molecules in a set of patients that received histotripsy, which is a non-invasive and non-thermal focused ultrasound therapy that induces mechanical stress to the cancerigenous cells, leading to its death (necrosis). Here we reported a focal decrease of the expression of these checkpoint molecules in tissue sections obtain at the treatment interface, compared to those taken from untreated areas of the tumor. In addition, a positive relationship was noticed between the infiltration of T lymphocytes and the expression of these checkpoint molecules in both canine melanoma, and soft tissue sarcoma. Our findings demonstrate that immunotherapies targeting these checkpoint molecules have a great potential for efficacy in canine neoplasms, along or combined with tumor ablation therapies that increased immune cell infiltration in poorly immunogenic neoplasia.

INTRODUÇÃO: Carcinomas mamários triplo-negativos correspondem ao grupo heterogêneo de neoplasias mamárias caracterizadas pela ausência de expressão dos receptores de estrogênio e progesterona e sem amplificação ou superexpressão do HER2. São mais prevalentes em mulheres jovens e em afrodescendentes. Eles se associam frequentemente ao fenótipo basal-símile determinado geneticamente, entretanto, incluem também outros tipos moleculares intrínsecos. Metodologias de análise genética de novas gerações têm permitido sua estratificação em subgrupos distintos, o que justifica a heterogeneidade clínica deste grupo de neoplasias. A identificação desses subgrupos através de marcadores imunoistoquímicos de aplicação prática ainda é pouco explorada, embora seja uma ferramenta promissora na sua estratificação e determinação de alvos terapêuticos. OBJETIVOS: Nosso objetivo é explorar os perfis imunoistoquímicos dos carcinomas mamários triplo-negativos em mulheres com idade até 45 anos e investigar possíveis diferenças entre as cinco regiões geográficas brasileiras. MÉTODOS: Selecionamos 118 amostras de tumores de pacientes com idade até 45 anos, com carcinoma invasivo, blocos de parafina disponíveis e perfil imunoistoquímico triplo-negativo, procedentes das cinco regiões geográficas. Estes casos foram revisados quanto à determinação de tipo e grau histológico e as seguintes características anatomopatológicas: contorno do tumor, presença e fração do componente \"in situ\", embolização vascular peritumoral, tipo e grau da reação estromal, presença de necrose tumoral e formação de túbulos pela neoplasia. Foram selecionadas áreas representativas do tumor para construção de blocos de microarranjos de tecido para estudo imunoistoquímico. Foram pesquisados os seguintes marcadores: citoqueratinas basais 5/6 e 14, citoqueratinas luminais 8 e 18, receptor do fator de crescimento epidérmico (EGFR ou HER1), receptor de androgênio, e-caderina, catenina-beta, claudinas 3, 4 e 7, vimentina, actina de músculo liso, p63, ALDH1 e Ki-67. De acordo com a expressão dos marcadores, os tumores foram classificados nos subgrupos basal-símile (expressão de citoqueratina basal 5/6 e/ou EGFR-positivo), claudina-baixo (claudinas e e-caderina-negativos), mesenquimal (vimentina-positivo), apócrino (receptor de androgênio-positivo), mioepitelial (p63 ou actina de músculo liso positivos), com perfil de células tronco (ALDH1 positivo), ou indefinido (padrões negativo para todos os marcadores). Nestes subtipos, a atividade proliferativa foi analisada através da expressão de Ki-67. As neoplasias provenientes das cinco regiões geográficas foram comparadas quanto às características histológicas e perfis imunoistoquímicos. RESULTADOS: A idade das pacientes variou de 26 anos a 45 anos ( média 38,3 +/-4,9 e mediana de 39 anos ). O tipo histológico mais frequente foi o carcinoma de tipo histológico não especial (107/118 casos; 90,7%). Observamos maior proporção de carcinomas de alto grau nas regiões nordeste, sul e sudeste. Os marcadores imunoistoquímicos não mostraram diferenças nas frequências entre as diferentes regiões geográficas. Observamos baixa atividade proliferativa determinada pela expressão do Ki-67 nos subgrupos com expressão do receptor de androgênio (apócrino) e sem expressão de vimentina (padrão não mesenquimal). Os tumores da região Nordeste, Sul e Sudeste apresentaram maior atividade proliferativa. Tumores ricos em linfócitos intratumorais apresentaram menor expressão de marcadores basais e do perfil basal-símile. CONCLUSÕES: Os subtipos moleculares determinados através da expressão imunoistoquímica não mostraram diferenças nas frequências entre as diferentes regiões geográficas. Os tumores das regiões Nordeste, Sul e Sudeste apresentaram maior atividade proliferativa. Os carcinomas ricos em linfócitos intratumorais apresentaram frequência menor do perfil basal / BACKGROUND: Triple-negative breast carcinomas (TNBC) correspond to a heterogeneous group of neoplasia characterized by the lack of expression of estrogen and progesterone receptors, and by the absence of amplification or overexpression of HER2. They are more prevalent among African descendants and younger women. They are often associated with the basal-like genetic phenotype; however, other intrinsic molecular types are included. Genomic analyses of next-generation methods have allowed stratification of TN breast carcinomas into distinct subgroups, explaining the clinical heterogeneity of this group of neoplasias. The identification of these subgroups by immunohistochemical markers is not well explored, although it represents a potential useful tool for the stratification and determination of therapeutic targets. OBJECTIVES: Our aim is to explore the TNBC immunohistochemical profile in patients of 45 year-old or younger, and to investigate possible differences among the five Brazilian geographic regions. METHODS: We\'ve selected 118 samples of tumors from patients up to 45 years-old from five Brazilian geographic regions, with invasive carcinoma, available paraffin blocks, and triple-negative immunohistochemical profile. All the cases were reviewed as for determination of histologic type and grading, and the following pathological features: tumor contour, presence and percentage of in situ component, peritumoral vascular embolization, type and grade of stromal reaction, presence of tumoral necrosis, and tubule formation by neoplasia. Representative tumor areas were selected for tissue microarray construction for immunohistochemical study. The following markers were studied: Basal cytokeratins 5/6 and 14; luminal cytokeratins 8 and 18; Epidermal growth factor receptor (EGFR or HER1); androgen receptor; e-cadherin; catenin-beta; claudins (3,4 and 7); vimentin; smooth-muscle actin; p63, ALDH1, and Ki-67. According to the expression of the markers, the tumors were grouped in the basal-like subgroups (basal cytokeratins 5/6 and/or EGFR positive), claudin-low (claudins and/or e-cadherin negative), mesenchymal (vimentin-positive), apocrine (androgen receptor positive), myoepithelial (p63 and/or smooth muscle actin positive), stem-cell (ALDH1-positive), or undefined (negative for all markers). In these subtypes the proliferative activity through the Ki-67 expression was analyzed. Neoplasias from the five regions were compared as for histological characteristics and immunohistochemical profiles. RESULTS: The age of patients ranged from 26 years to 45 years (mean 38.3 +/- 4.9 and median of 39 years). The most common histological type was no special histological type (NST) of carcinoma (107/118 cases, 90.7%). We observed a higher proportion of high-grade carcinomas in the regions Northeast, South and Southeast. Compared to the Midwestern and Northern regions there was no statistically significant difference (p = 0.03). The immunohistochemical markers showed no differences in the frequencies between the different geographical regions. We observed low proliferative activity determined by Ki-67 expression in the subgroups with androgen receptor expression (apocrine) and no expression of vimentin (no mesenchymal pattern). Tumors of the Northeast, South and Southeast have higher proliferative activity. There was a lower frequency of immunohistochemical markers associated with basal molecular type between tumors rich in lymphocytes. CONCLUSIONS: The molecular subtypes determined by immunohistochemical expression have shown no differences in the frequencies among the different geographical regions. Tumors in the Northeast, South and Southeast had higher proliferative activity. Carcinomas rich in intratumoral lymphocytes had lower frequency of basal profile