Yes / Background: miRNAs (microRNAs) play a key role in triple-negative breast cancer (TNBC) progression, and its heterogeneity at the expression, pathological and clinical levels. Stratification of breast cancer subtypes on the basis of genomics and transcriptomics profiling, along with the known biomarkers’ receptor status, has revealed the existence of subgroups known to have diverse clinical outcomes. Recently, several studies have analysed expression profiles of matched mRNA and miRNA to investigate the underlying heterogeneity of TNBC and the potential role of miRNA as a biomarker within cancers. However, the miRNA-mRNA regulatory network within TNBC has yet to be understood. Results and Findings: We performed model-based integrated analysis of miRNA and mRNA expression profiles on breast cancer, primarily focusing on triple-negative, to identify subtype-specific signatures involved in oncogenic pathways and their potential role in patient survival outcome. Using univariate and multivariate Cox analysis, we identified 25 unique miRNAs associated with the prognosis of overall survival (OS) and distant metastases-free survival (DMFS) with “risky” and “protective” outcomes. The association of these prognostic miRNAs with subtype-specific mRNA genes was established to investigate their potential regulatory role in the canonical pathways using anti-correlation analysis. The analysis showed that miRNAs contribute to the positive regulation of known breast cancer driver genes as well as the activation of respective oncogenic pathway during disease formation. Further analysis on the “risk associated” miRNAs group revealed significant regulation of critical pathways such as cell growth, voltage-gated ion channel function, ion transport and cell-to-cell signalling. Conclusion: The study findings provide new insights into the potential role of miRNAs in TNBC disease progression through the activation of key oncogenic pathways. The results showed previously unreported subtype-specific prognostic miRNAs associated with clinical outcome that may be used for further clinical evaluation. / EPSRC (EP/R043787/1).

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-09-04T15:39:47Z No. of bitstreams: 1 homerogoncalvesjunior.pdf: 2526505 bytes, checksum: e3bc0f5bc2176febea8072cc318baab6 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-09-04T15:52:35Z (GMT) No. of bitstreams: 1 homerogoncalvesjunior.pdf: 2526505 bytes, checksum: e3bc0f5bc2176febea8072cc318baab6 (MD5) / Made available in DSpace on 2018-09-04T15:52:35Z (GMT). No. of bitstreams: 1 homerogoncalvesjunior.pdf: 2526505 bytes, checksum: e3bc0f5bc2176febea8072cc318baab6 (MD5) Previous issue date: 2018-07-06 / O tratamento do câncer de mama baseia-se na classificação dos casos, em termos de estadiamento e do perfil biomolecular. Os Tumores Triplo Negativos (TTN) representam um grupo especial de neoplasias mamárias que não expressam receptores hormonais e nem o antígeno Her2. São considerados agressivos e de pior evolução, e quando estudados em particular, apresentam muita heterogeneidade. Importa saber se a caracterização dos tumores como Triplo Negativos, é suficiente para delimitar o grupo em termos de prognóstico e terapêutica. Este estudo teve como objetivo comparar os aspectos clínico-epidemiológicos dos Tumores Triplo Negativos em relação aos Não Triplo Negativos, em coorte de mulheres com câncer de mama assistidas em centros oncológicos de referência de Juiz de Fora, Minas Gerais. A sobrevida global e a sobrevida livre de doença foram calculadas pelo método de Kaplan Meier, e as curvas de sobrevida foram avaliadas pelo teste de Log-Rank, nos subgrupos Triplo Negativos e Não Triplo Negativos (NTN). Os fatores prognósticos foram comparados pelo modelo de riscos proporcionais de Cox. Os Tumores Triplo Negativos apresentaram diferenças demográficas em relação aos NTN, com acúmulo de pacientes não brancas e de baixo nível sociocultural; e ainda com aspectos de maior gravidade ao diagnóstico. A evolução também foi pior, tanto em termos de sobrevida global quanto sobrevida livre de doença dentre os TTN. Na análise univariada, os fatores: idade, cor da pele, escolaridade, tamanho do tumor e grau tumoral, estado das axilas e estadiamento, bem como taxas elevadas dos marcadores P53 e Ki 67, se mostraram associados a sobrevida livre de doença nos Tumores Não Triplo Negativos. No cálculo da sobrevida global, essas variáveis se mantiveram, exceto a idade; e foi constatado maior risco para as mulheres oriundas do serviço público de saúde, bem como o surgimento de metástases no decurso do seguimento. Para os Triplo Negativos, a análise univariada mostrou influência do estado axilar e estadiamento na sobrevida livre de doença; e os mesmos fatores acrescidos do surgimento de metástases, para a sobrevida global. Na análise multivariada a escolaridade e o estado axilar representaram risco à sobrevida livre de doença para NTN, enquanto a cor da pele e o estadiamento para a sobrevida global. Quanto aos TTN, sua evolução se mostrou ligada a dois aspectos: o comprometimento axilar para sobrevida livre de doença e global; e também a multicentricidade para a sobrevida global. Os Tumores Triplo Negativos aparentam ter biologia bem diversa dos Não Triplo Negativos, na dependência dos componentes histológicos e moleculares que portam. A classificação molecular por imunoistoquímica se mostrou capaz de identificar os dois grupos tumorais e auxiliar na orientação terapêutica. / Current breast cancer treatment is based on the classification of tumor stage and molecular profile. Triple-negative breast cancer (TNBC) is a specific subset of tumors characterized by the absence of hormone and HER2 receptors. Despite being usually associated with a more aggressive clinical course, there is high heterogeneity within TNBC. Therefore, it has been questioned whether current classification of TNBC is adequate enough to assess its prognosis and make therapeutic decisions. This study thus aimed to investigate to which extent TNBC profile classification was able to efficiently distinguish this tumor subtype from other subtypes of breast cancer. It was performed on a cohort of women with breast cancer treated at referral centers in Juiz de Fora, Southeastern Brazil. Overall and disease-free survival and prognostic factors were assessed and compared for TNBC and non-TNBC. Survival functions were calculated using the Kaplan-Meier method, and the log-rank test was used to compare the survival curves. Prognostic factors were analyzed by the Cox proportional hazards model. TNBC presented demographic differences compared to non-TNBC as it was more prevalent among nonwhite and less educated women. TNBC also presented at diagnosis with clinical parameters of advanced disease and had overall and disease-free survival significantly lower than non-TNBC. In univariate analysis the factors: age, color of the skin, education level, size and degree of tumor, axillary status and staging, as well as high rates of P53 e Ki 67 have been shown to be associated with disease-free survival in non-TNBC. These variables remained the same in the calculation of overall survival except for age; and it was also observed a greater risk for women from the public health service as well as the appearance of metastases during the follow-up. In multivariate analysis education level and axillary lymph node involvement presented a risk for disease-free survival while the color of skin and staging, for overall survival in non-TNBC. Regarding TNBC, its evolution was related to two aspects: axillary impairment for disease-free and global survival and multicentricity for overall survival. TNBC presents distinct biological properties compared to non-TNBC, which seems to be related to its specific histological and molecular components. The molecular classification by immunohistochemistry showed to be able to identify the two tumor groups and to support the therapeutic orientation.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in the majority of triple negative breast cancer patients (TNBC). However, the molecular determinants behind their limited response to EGFR-targeted therapies are poorly understood. Here, both the acute and chronic responses of TNBC to the EGFR-targeted therapy, cetuximab (CTX), have been investigated. The expression of EGFR has been analyzed in a cohort of 2000 breast cancer tumours from the public dataset as well as in a panel of breast cancer cell lines. Furthermore, the response of TNBC cell lines to CTX has been investigated using conventional biochemical methods. Finally, a comprehensive transcriptomic profiling of an acquired CTX-resistant TNBC model by RNA sequencing has been performed to understand the molecular determinants of acquired CTX resistance. The results confirmed that EGFR is highly expressed in TNBC in comparison to non-TNBC breast cancer tumours and cell lines, which was associated with adverse clinical outcomes. Targeting EGFR in TNBC cell lines using CTX failed to completely inhibit the EGFR signalling pathway and was associated with an increase in ADAMs-mediated release of endogenous EGFR ligands, EGF and TGFα. Inhibition of ADAMs (ADAM10 and ADAM17) significantly enhanced the anti tumour efficacy of CTX both in vitro and in vivo. Furthermore, transcriptomic profiling of the acquired CTX-resistant TNBC cell line (MDA-MB-468CR) revealed an activation of several key oncogenic pathways and genes, including the TGFβ/BMP pathway. Blocking BMP receptors (BMPRs) restored the sensitivity of resistant cells to CTX treatment. Collectively, current findings offer alternative strategies that could enhance the CTX response in TNBC. We further reported that simultaneous targeting of both EGFR and BMPR pathways could overcome CTX resistance, which might have important implications for the treatment of TNBC.

La transition épithélio-mésenchymateuse (EMT) confère un phénotype agressif aux cancers du sein avec la perte de leurs jonctions épithéliales intercellulaires, leur polarité et identité, et l’acquisition des caractéristiques mésenchymateuses accompagnées de l’invasion et la résistance à la chimiothérapie. Cependant, les mécanismes et les signaux associés qui régissent ces dysfonctionnements au cours de l’EMT des cancers du sein demeurent encore mal connus. Dans cette étude, nous avons entrepris de comparer deux modèles génétiques des cellules MCF-7 du cancer du sein en EMT induite par l’expression constitutive de Snail ou l’invalidation de WISP-2. Nous avons trouvé que les répresseurs des E-cadhérines tels que Slug, Zeb1/2, Twist et Snail étaient surexprimés dans les cellules en EMT avec l’induction d’un phénotype triple-négatif (TN), la perte du récepteur aux oestrogènes, GPR30, et le gain du pouvoir invasif dans le collagène de type I. De plus,l’expression ectopique de Snail dans les cellules transfectées supprime aussi les transcrits de WISP-2. L’EMT est accompagnée de la dominance de plusieurs voies de signalisation proinvasives impliquant les protéines GαGβγ, PKCα, AKT et l’induction de c-Jun. Nous avons mis en évidence l’activation du cycle cellulaire et l’inhibition de l’axe p27/cyclindependent kinase CDK3 responsable de la promotion de la croissance cellulaire. De manière intéressante, le traitement des cellules en EMT avec les inhibiteurs des GαGβγ (BIM-46187, Gallein), PKCα (Gö6976, MT477, sh-RNAs) et de la cascade de signalisation PI3K/AKT (wortmannin) réduit leur potentiel invasif. Par contre, les inhibiteurs sélectifs des récepteurs HER et des voies de signalisation MAPK/SAPK n’ont aucun effet sur les deux modèles d’EMT signant leur indépendance à ces voies oncogéniques. L’ensemble de nos résultats suggèrent que les protagonistes de signalisation représentés par les GαGβγ, PKCα et PI3K/AKT peuvent être considérés comme de potentiels biomarqueurs prédictifs et des cibles thérapeutiques dans la prise en charge des cancers du sein TN en EMT. / The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype to breast cancers (BC) following disruption of intercellular junctions, epithelial cell polarity, induction of mesenchymal traits, invasive growth and chemotherapy resistance. However, the mechanisms underlying the EMT and its associated signaling dysfunctions in BC are still poorly understood. Here, we have undertaken a comparative study in two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation.We report that the E-cadherin repressors Slug, Zeb1/2 and Twist were overexpressed in these EMT cells. They induced a triple negative phenotype (loss of estrogen ERα and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the GPR30 ER and promotion of invasive growth in collagen gels. Ectopic Snail expression suppressed WISP-2 transcripts and down-regulated WISP-2 gene promoter expression in transfected cells. The EMT caused dominance of several proinvasive pathways including GαGβγ subunits, PKCα, AKT and c-Jun induction, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27kip1/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting GαGβγ subunits (BIM-46187, gallein), PKCα (Gö6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin) alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signalingindependence to inhibitors of HER family tyrosine kinases and the mitogen- and stressactivated protein kinases. Our study suggests that the signaling protagonists GαGβγ, PKCα and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the management of clinical BC in EMT.

Made available in DSpace on 2016-12-23T13:49:03Z (GMT). No. of bitstreams: 1 Isabella dos Santos Guimaraes.pdf: 1486152 bytes, checksum: 65f12f2a8d639d0b762766b845fea774 (MD5) Previous issue date: 2013-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Triple-negative breast cancer (TNBC) is a heterogeneous and poor prognostic disease, partially due to its ineligibility to target therapy, and the high relapse index, despite the initial satisfactory response to antineoplastic drugs. Therefore, the identification of alternative substances is urgent, as herein presented with regard to metformina and TNBC (lineage MDAMB231). Despite the low percentage of apoptotic/necrotic cells induced by metformin, 4.1% (10 μM) e 8.8% (54 mM), there was a discrete increase of cells in sub-G0, which is in agreement with the low value, despite doubled, of apoptosis/necrosis induced by metformina 54 mM. Metformin reverted ERK constitutive activation, possible antineoplastic mechanism; however, only reduced cellular proliferation at high doses (10 μM vs. 54 mM, 9.8% vs. 45.23%, respectively), possibly due to the modulation of other mechanisms of action. Paclitaxel 0.25 μM induced apoptosis/necrosis in 29% of the cells, which is comparable to the 36.34% of dead cells obtained by MTT. Despite the cell cycle arrest in G2/M that favours its mechanism of action, its antineoplastic efficacy can be compromised by ERK constitutive activation, which is related to taxane chemoresistance. Metformin 10 μM combined with paclitaxel 0,25 μM, although synergic with regard to the antineoplastic effect (dead cells = 53.20%), kept constant the induction of apoptosis/necrosis when compared to paclitaxel alone, as the benefit of arresting the cell cycle at G2/M can be overcome by ERK constitutive activation. When administered at their IC50, metformin 54 mM and paclitaxel 0,25 μM, the percentage of apoptotic/necrotic cells increased to 41%, which is comparable to the decrease in cell proliferation (59.9% of dead cells). Worthwhile pointing that: i) ERK constitutive activation was reversed, favoring paclitaxel toxicity; ii) the number of cells in sub-G0 increased. Indeed, among the 41% apoptotic/necrotic cells, 21.4% derived from late apoptosis, thus justifying sub-G0 expansion. The data point to mutual synergism between metformina and paclitaxel. Our findings support that, among other mechanisms of action, metformina is an indirect inhibitor of ERK, possibly of mTOR as well, explaining, therefore, its potential application in the treatment of TNBC, a major challenge of clinical oncology / O câncer de mama triplo-negativo (TNBC) configura doença heterogênea e de prognóstico ruim, dentre outras razões, por não ser responsivo às terapias-alvo e pelo elevado índice de relapso da doença apesar da responsividade inicial satisfatória aos antineoplásicos. Sendo assim, há urgência na identificação de substâncias alternativas, como ora apresentado em relação à metformina e TNBC (linhagem MDAMB231). Embora a porcentagem total de células em apoptose/necrose induzida por metformina seja pequena, 4,1% (10 μM) e 8,8% (54 mM), houve aumento discreto de células na fase sub-G0; aspecto concordante com o valor baixo, ainda que dobrado, de indução apoptose/necrose por metformina 54 mM. Metformina reverteu a ativação constitutiva de ERK, possível mecanismo antineoplásico da droga, mas somente reduziu a proliferação celular em doses elevadas (10 μM vs. 54 mM, 9,8% vs. 45,23%, respectivamente), possivelmente pela modulação de outros mecanismos de ação. Paclitaxel 0,25 μM promoveu apoptose/necrose em 29% das células, valor compatível os 36,34% de células mortas por MTT. Apesar da parada do ciclo celular em G2/M, favorecendo seu mecanismo de ação, sua eficácia antineoplásica limitada pode decorrer da sustentação da ativação constitutiva de ERK, já relacionada à quimiorresistência ao taxano. A combinação de metformina 10 μM com paclitaxel 0,25 μM, embora seja sinérgica quanto à ação antineoplásica (células mortas = 53,20%), manteve constante a indução de apoptose/necrose quando comparado ao paclitaxel sozinho, já que o benefício do aumento de células em G2/M pode ter sido sobreposto pela ativação de ERK. Quando administradas em suas IC50, metformina 54 mM e paclitaxel 0,25 μM, o percentual total de células apoptóticas/necróticas aumentou para 41%, valor mais próximo àquele de diminuição da proliferação celular (59,9% de células mortas). É notório ter havido: i) reversão da ativação constitutiva de ERK, favorecendo a citotoxicidade de paclitaxel; ii) aumento de células em sub-G0. De fato, dos 41% de células em apoptose/necrose, 21,4% derivaram de efeito apoptótico tardio, justificando a expansão de sub-G0. Os dados apontam para sinergismo mútuo entre metformina e paclitaxel. Nossos achados evidenciam que, dentre outros possíveis mecanismos, a metformina atua como inibidor indireto de ERK, possivelmente também de mTOR, explicando, portanto, seu uso potencial no tratamento do TNBC, um dos grandes desafios da clínica oncológica

No description available.

Poly-(ADP-ribsoyl) Polymerases (PARPs) are a superfamily of enzymes comprised of 17 known isoforms. PARP inhibitors (PARPi) have shown success in clinical trials for the treatment of homologous recombination-deficient cancers. Though proven effective initially, tumors treated with PARPi eventually develop resistance. Combinatorial therapeutics targeting PARP and other pathways that may re-sensitize tumors to PARP inhibition, including PI3K/AKT/mTor pathway, and cell-cycle checkpoints (such as CDKs, CHK, and Wee) are being tested. In this context, the synthetic lethality of cyclin-dependent kinase 1 (CDK1) and PARP1 is known. Evaluation of PARP1 and CDK1 pharmacophores led to the development of the tetrahydro-arylazepinone (TAAP) scaffold as a potential dual PARP1/CDK1 inhibitor. We screened a handful of TAAP analogs against PARP1 in a cell-free assay that identified the low micromolar PARP1 inhibitor 1,2,3,4-tetrahydro-5H-benzo[e][1,4]-diazepin-5-one (TBAP), which served as the lead compound. The analogous 1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]-diazepin-5-one (TPAP) series showed a similar bioactivity profile. Satisfyingly, the N1-benzyl TPAP analogue showed activity in the low nanomolar range. The TAAP series (i.e., 6/7-membered scaffold) unfortunately lacked CDK1 inhibitory activity. Finally, many PARPi's show poor isoform-selectivity. The development of isoform-selective PARPi can clarify the specific function of each PARP isoform and may reduce the adverse side effects shown by PARPi. A handful of TAAP analogs were screened against 13 PARP isoforms, where some compounds demonstrated exquisite PARP1/2 selectivity. Concurrently, we discovered an inhibitor for PARP11, an isoform that lacks any known synthetic ligand. Future directions are suggested towards fine-tuning the structure-activity relationship of TAAP-isoform selective PARPi as well as developing a dual PARP1/CDK1 inhibitor. / Master of Science

Les cellules souches cancéreuses (CSC) sont à l’origine de la progression tumorale, des métastases et rechutes tardives. Elles ont été identifiées dans de nombreux cancers, comme le cancer du sein triple négatif (TNBC) et cancers de grade III-IV. Elles sont résistantes aux chimiothérapies et radiothérapie et résident dans une niche immuno-répressive. Cette étude vise à évaluer une stratégie d’immunothérapie qui cible sélectivement les CSC dans le modèle murin 4T1-GFP-Luc mimant le TNBC. Le phénotype/ génotype des mamosphères a été initialement caractérisé. Basée sur l’analyse génomique des CSC, nous avons développé une immunothérapie active associée à des agents immuno-modulateurs. Nous avons mesuré la taille des tumeurs et suivi l’apparition des métastases par bioluminescence. Une étude immunologique et analyse génomique de la tumeur a été réalisée. La combinaison thérapeutique provoque le recrutement dans la tumeur de lymphocytes T (CD4 +, CD8 +) et lymphocytes B par augmentation de CXCL13, une réduction des lymphocytes T reg et cellules myéloïdes suppressives. Cette induction de réponse immunitaire provoque la diminution de la taille de la tumeur et des métastases. Cette nouvelle immunothérapie active de type vaccinale pourra être utilisée en association avec les traitements actuels pour des mesures prophylactiques et curatives dans une grande variété de cancers. / Cancer stem cells (CSCs) are responsible for tumor progression, metastases, and late relapses. They have been identified in many cancers, such as triple negative breast cancer (TNBC) and grade III to IV cancers. They are resistant to chemotherapy and radiotherapy and reside in an immuno-repressive niche.This study aims to evaluate a immunotherapy strategy that selectively targets CSCs in the mouse model 4T1-GFP-Luc mimicking TNBC. The phenotype / genotype of mammosphere was initially characterized. Based on genomic analysis of CSC, we have developed an active immunotherapy associated with immunomodulatory agents. We measured the size of tumors and monitored the appearance of metastases by bioluminescence. We performed an immunological study and genomic tumor analysis. The therapeutic combination causes the recruitment of CD4 + and CD8 + T lymphocytes and B lymphocytes with increased CXCL13, the reduction of T reg cells and suppressive myeloid cells in the tumor. This induction of intra-tumor immune response leads to a decrease in tumor size and metastases.This new active immunotherapy can be used in combination with current treatments for prophylactic and curative measures in a wide variety of cancers.

Le cancer du sein triple négatif (CSTN) est le sous-type de cancer du sein le plus hétérogène et le plus défavorable. La pierre angulaire du traitement de ces tumeurs repose sur la chimiothérapie systémique, de plus en plus fréquemment administrée en néoadjuvant, puisqu’aucune thérapie ciblée n’est à ce jour validée. L'obtention d'une réponse complète histologique (RCH) constitue un marqueur pronostique favorable majeur ainsi qu'un test in vivo de la sensibilité aux médicaments anti-tumoraux. L’objectif de notre travail de thèse a donc été d’apporter des éléments de compréhension de cette hétérogénéité grâce à la dissection clinique, biologique et moléculaire de ces tumeurs. Nous avons analysé les profils d'expression géniques de ces CSTN et ainsi identifié 6 sous-types moléculaires distincts avec des biologies et des pronostics différents. Cette classification s’appuie sur une méthodologie originale basée à la fois sur des outils bioinformatiques classiques associée à l’utilisation de réseaux biologiques. L’enrichissement en gènes de l'immunité issus du compartiment stromal de la tumeur représente un déterminant majeur du pronostic de ces tumeurs : une forte expression des gènes de l'immunité est associée un pronostic significativement plus favorable. Notre principale contribution repose sur une meilleure compréhension de l’immunité et de l’infiltrat lymphocytaire (TILS) de ces CSTN. Il s’agit probablement du sous-groupe de cancers du sein le plus immunogène avec des taux de TILS pré-CNA parmi les plus élevés avec les tumeurs HER2-positives. Cet infiltrat lymphocytaire est d'ailleurs très corrélé aux gènes de notre module immunitaire pronostique dans les CSTN. La valeur prédictive et pronostique des TILS du stroma tumoral est différente selon le sous-type moléculaire de cancer du sein, suggérant une immunité complètement différente de ces tumeurs. Le taux de TILS varie également différentiellement au sein de chaque sous-groupe sous l'influence de la CNA, témoignant d'une interaction complexe entre les TILS et les traitements. Nous montrons que la cinétique des TILS sous l'effet de la CNA est un indicateur pertinent de réponse à la CNA avec une réponse d'autant plus importante qu'une décroissance du taux de TILS sera importante. Les tumeurs les plus immunogènes avec une activité immunitaire importante sont donc les tumeurs triple-négatives les plus favorables. L'un des challenge des années à venir sera donc d'identifier le plus tôt possible les CSTN les moins immunogènes susceptibles de bénéficier au mieux des immunothérapies seules ou combinées au traitement chimiothérapique afin d'activer ou de rétablir précocement une immunité déficiente. Sous une même dénomination de TILS se trouve très certainement des populations phénotypiques de lymphocytes différentes. En effet, après CNA, leur valeur pronostique est opposée entre les CSTN et les tumeurs HER2-positives: des taux élevés de TILS sont associés à un pronostic défavorable dans les tumeurs HER2-positives alors qu’ils ont une tendance à être associés à des CSTN de meilleur pronostic. Les interactions sont complexes entre les agents cytotoxiques et la tumeur et/ou son microenvironnement. L'analyse du résidu tumoral mammaire ou ganglionnaire représente un matériel sous-exploité qui pourrait permettre de mieux comprendre les mécanismes de sensibilité ou de résistance aux traitements. Au delà de la pierre angulaire que constitue l'immunité pour ces tumeurs, nos travaux identifient certains CSTN pour lesquels l'environnement hormonal, au travers de l'indice de masse corporel ou du statut ménopausique, pourrait jouer un rôle. Ainsi l'exploration du métabolome, des particularités immunitaires chez les patientes en surpoids/obèses ou l'analyse de la voie androgène-récepteur (et ses connexions avec les voies oestrogène et progestérone-récepteur) des CSTN doit aussi être explorée de manière détaillée. Ceci ouvre des perspectives de traitement possibles pour certaines patientes. / Triple negative breast cancer (TNBC) is the most heterogeneous and pejorative subtype of breast cancer. The cornerstone of the treatment of such tumors is based on systemic chemotherapy, more and more frequently administered before surgery, since no targeted therapy has been validated to date. Obtaining a pathological complete response (PCR) is a major favorable prognostic marker as well as an in vivo test of anti-tumor drug susceptibility. The objective of our thesis work was therefore to provide elements of understanding of this heterogeneity through the clinical, biological and molecular dissection of these tumors.We analyzed gene expression profiles of TNBCs and identified 6 distinct molecular subtypes with different biologies and prognoses. This classification used an original methodology based on both classical bioinformatic tools and biological networks. The enrichment of immunity genes from the stromal compartment of the tumor represents a major determinant of the prognosis of these tumors: a strong expression of the immunity genes is associated with a significantly more favorable prognosis.Our main contribution is based on a better understanding of immunity and tumor infiltrated lymphocytes (TILS) of these TNBCs. It is probably the most immunogenic subgroup of breast cancers with the highest TILs levels pre-NAC with HER2-positive tumors. TILS levels are also highly correlated with genes of our immune prognosis module. The predictive and prognostic value of stromal TILS is different according to the molecular subtype of breast cancer, suggesting a completely different immunity of these tumors. The rate of TILS also varies differentially within each subgroup under the influence of the NAC, indicating a complex interaction between TILS and treatments. We show that the kinetics of TILS levels with NAC is a relevant indicator of response to NAC with a response that is all the more important that a decrease in the TILS rate will be important. The most immunogenic tumors with an important immune activity are therefore the most favorable triple-negative tumors. One of the challenges of the coming years will therefore be to identify, as soon as possible, the least immunogenic TNBC that can best benefit from immunotherapies alone or in combination with chemotherapeutic treatment in order to activate or restore early a deficient immunity.Under the same denomination of TILS there are probably different phenotypic populations of lymphocytes. Indeed, after CNA, their prognostic value is opposite between the TNBC and the HER2-positive tumors: high levels of TILS are associated with an unfavorable prognosis in HER2-positive tumors whereas they have a tendency to be associated with a better prognosis for TNBC tumors. The interactions are complex between cytotoxic agents and the tumor and / or its microenvironment. The analysis of the breast or axillary lymph node residual disease represents an underutilized material that could lead to a better understanding of the mechanisms of sensitivity or resistance to treatment.Beyond the key role immunity for these tumors, our work identifies some TNBCs for which the hormonal environment, through the body mass index or the menopausal status, could play a role. Thus, the exploration of the metabolome, immune features in overweight / obese patients or the analysis of the androgen-receptor pathway (and its connections with the estrogen and progesterone-receptor pathways) of the TNBC must also be explored. This opens up possible treatment perspectives for some patients.

Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancers (TNBCs) are pathologically defined by the absence of estrogen, progesterone, and HER2 receptors. Compared to other breast cancers, TNBC has a relatively high mortality. In addition, TNBC patients are more likely to relapse in the first few years after treatment, and experiencing a shorter median time from recurrence to death. Detecting the presence of tumor in patients who are technically “disease-free” after neoadjuvant chemotherapy and surgery as early as possible might be able to predict recurrence of patients, and then provide timely intervention for additional therapy. To this end, I applied the analysis of “liquid biopsies” for early detection of minimal residual disease (MRD) on early-stage TNBC patients using next-generation sequencing. For the first part of this study, I focused on detecting circulating tumor DNA (ctDNA) from TNBC patients after neoadjuvant chemotherapy and surgery. First, patient-specific somatic mutations were identified by sequencing primary tumors. From these data, 82% of the patients had at least one TP53 mutation, followed by 16% of the patients having at least one PIK3CA mutation. Next, I sequenced matched plasma samples collected after surgery to identify ctDNA with the same mutations. I observed that by detecting corresponding ctDNA I was able to predict rapid recurrence, but not distant recurrence. To increase the sensitivity of MRD detection, in the second part I developed a strategy to co-detect ctDNA along with circulating tumor RNA (ctRNA). An advantage of ctRNA is its active release into the circulation from living cancer cells. Preliminary data showed that more mutant molecules were identified after incorporating ctRNA with ctDNA detection in a metastatic breast cancer setting. A validation study in early-stage TNBC is in progress. In summary, my study suggests that co-detection of ctDNA and ctRNA could be a potential solution for the early detection of disease recurrence. / 2021-08-05