La chirurgie est la pierre angulaire du traitement curatif des sarcomes, lorsqu’elle est possible. En revanche, en cas de maladie avancée ou métastatique, les traitements systémiques ont une efficacité assez limitée avec un réel besoin de nouvelles options thérapeutiques. Le récent succès de l’immunothérapie dans les tumeurs épithéliales soulève donc la question de la possibilité d’une telle approche dans les sarcomes, et surtout pour quels sous-types histologiques. L’objectif de ce travail de thèse était d’obtenir des données précliniques en caractérisant le microenvironnement immunitaire au sein de trois types de sarcomes potentiellement candidats à l’immunothérapie, prérequis indispensable avant d’envisager une application clinique : 1) Dans le chondrosarcome, l’expression de PD-L1 a été retrouvée exclusivement dans près de 50% des chondrosarcomes dédifférenciés, et s’associait à une infiltration lymphocytaire T et l’expression des molécules HLA de classe I. Ces données incitent donc à inclure les patients avec ce sous type de chondrosarcome dans des essais cliniques évaluant un traitement anti PD-1/PD-L1. 2) Dans l’ostéosarcome, un infiltrat lymphocytaire T était observé de façon bien plus importante dans les lésions métastatiques que dans lésions primitives ou rechutes locales. De plus, l’expression de PD-L1 était retrouvée dans presque 50% des métastases mais pas ou peu dans la tumeur primitive correspondante, traduisant ici une dynamique d’échappement au système immunitaire lors de la progression de la maladie. Une stratégie ciblée sur les lymphocytes T visant à amplifier et potentialiser cette réponse immune préexistante dans les lésions métastatiques pourrait donc offrir un bénéfice clinique. 3) Dans le léiomyosarcome, les molécules HLA de classe I étaient fortement exprimées et l’expression de PD-L1 retrouvée dans 30% des tumeurs de haut grade, également très infiltrées par des macrophages immunosuppresseurs CD163+. Une importante infiltration de macrophages CD163+ était un marqueur indépendant de mauvais pronostic pour la survie, indiquant l’intérêt de d’une approche ciblée visant les macrophages dans ce type de sarcome, éventuellement en association avec un traitement anti PD-1/PD-L1. / Local control with adequate surgery is the cornerstone of sarcoma treatment. However, most sarcoma lack effective systemic therapies in case of advanced disease, emphasizing an unmet medical need for new therapeutic targets. The recent success of immunotherapy in epithelial malignancies raises the question whether such therapies, and which ones, would be applicable in sarcomas. As a prerequisite for therapeutic applications, we characterized the immune microenvironment in three sarcoma subtypes potentially candidate to immunotherapy: 1) In chondrosarcoma, PD-L1 expression was exclusively found in nearly 50% of the dedifferentiated subtype, in association with immune-infiltrating cells and HLA class I expression. These data provide rationale for including such patients in clinical trials with PD-1/PD-L1-targeted therapies. 2) In osteosarcoma, we observed a high density of tumor-infiltrating T cells in metastatic lesions compared to primary tumors and local relapses. Furthermore, PD-L1 positivity in almost half of metastases while mainly negative in the associated primary tumors, emphasises the dynamics of an adaptive mechanism of immune escape. Enhancing the preexisting immune response in metastatic lesions using T-cell-based immunotherapy may offer clinical benefit. 3) In leiomyosarcoma, HLA class I molecules were strongly upregulated and PD-L1 expression found in 30% of high-grade tumors, which were also highly infiltrated with CD163+ immunosuppressive macrophages. CD163+ was found to be an independent poor prognostic factor for overall survival, indicating the need for assessing a macrophage-targeted approach in this tumor type, as single agent or in combination with anti PD-1/PD-L1agents.

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Precision medicine has allowed for the development of monoclonal antibodies that unmask the anti-tumor immune response. These agents have provided some patients durable clinical benefit. However, PD-1 and PD-L1 inhibitor therapies are effective in a small group (10-20%) of non-small cell lung cancer (NSCLC) patients when used as single-agent therapy. The approved companion diagnostic is expression of the immune cell surface molecule, programmed death ligand 1 (PD-L1), on tumors measured by immunohistochemistry (IHC). Studies in tumor biology and immune surveillance dictate that PD-1 inhibitor efficacy should depend on the level of PD-L1 expression; however, the literature has not followed with convincing evidence. The limitations of this test include timing of tissue acquisition, tumor heterogeneity, and timing of therapy relative to the expression of PD-L1. In addition, the requirement of analyzing tumor tissue biopsy samples from a patient is cumbersome. Thus, a peripheral blood biomarker that predicts efficacy of PD-1/PD-L1 inhibition would be optimal for precise and cost-effective treatment. A history of chronic inflammatory diseases may be advantageous for a cancer patient who is treated with PD-1/PD-L1 inhibitors and may allow them to then mobilize a swift immune response to tumor cells. Specific biological components of this persistent inflammation may predict PD-1 inhibitor response. We have taken a novel approach to leverage national healthcare claims data that couples patient history with response to therapy. We have identified potential peripheral blood biomarkers of response to PD-1/PD-L1 inhibitors using a combination of healthcare outcomes and molecular markers that correlate with therapeutic efficacy.

The immune checkpoint blockade is a novel approach of cancer therapy, which markedly enhanced treatment efficacy of several cancer types. However, the frequency of cancer patients non-responding to this treatment is high. Establishment of predictive markers to distinguish patients suitable for the immune checkpoint blockade would enhance the number of patients receiving benefit from the therapy. This dissertation thesis focuses on the enhancement of efficacy of immune checkpoint inhibitors (ICIs) and predictive markers in experimental models of mouse tumours induced by TC-1 and TC-1/A9 cell lines and its clones with deactivation of interferon (IFN)-γ signalling (TC-1/dIfngr1 and TC-1/A9/dIfngr1), or CD80 molecule (TC-1/dCD80-1). IFN-γ is presumed to be the main inducer of programmed death ligand 1 (PD-L1) and a major histocompatibility complex I (MHC-I). Moreover, PD-L1 expression may predict sensitivity to PD-1/PD-L1 blockade. Non-functional IFN-γ signalling or downregulated MHC-I expression has been associated with resistance to ICIs in some patients. We found that IFNs type I (IFN-α and IFN-β) induced the expression of PD-L1 and MHC-I on TC-1/A9/dIfngr1 tumour cells with reversible downregulation of both molecules. We also showed that deactivation of IFN-γ signalling in TC-1/A9 cells was not a...

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.

Le virus d’Epstein-Barr (EBV) est le premier virus transformant à avoir été identifié chez l’Homme. Il infecte plus de 90% de la population adulte mondiale, persistant sous forme épisomale dans le compartiment B mémoire tout au long de la vie de l’hôte. Lors de la primo-infection et lors de phases de réactivation du virus, les cellules B immortalisées sont en programme de latence III, aussi appelée phase de prolifération, où l’ensemble des protéines de latence sont exprimées. Lorsque les hôtes sont immunocompétents, un équilibre entre hôte et virus s’établit et la plupart des cellules B infectées sont éliminées par le système immunitaire de l’hôte, principalement par les lymphocytes T cytotoxiques. En cas de déficit immunitaire, il peut y avoir émergence de lymphomes, tels que les désordres lymphoprolifératifs des patients immunodéprimés (PTLDs), les lymphomes non-Hodgkiniens (LNH) et Hodgkiniens (LH). Les travaux antérieurs du laboratoire ont permis de révéler que l’immuno-inhibiteur PD-L1/B7-H1/CD274 est surexprimé à la surface des lymphocytes B en latence III de l’EBV. L’interleukine-10 (IL-10) est également sécrétée par ces cellules. Ces caractéristiques sont communes aux cellules B régulatrices (Bregs). Le but de ma thèse était d’interroger les caractéristiques immuno-modulatrices des cellules B en latence III de l’EBV, dans le cadre des propriétés des Bregs. Nous montrons que les cellules B en latence III de l’EBV possèdent les déterminants antigéniques communs aux Bregs immatures (CD24High CD38High PD-L1High), associée à une surexpression des cytokines immunosuppressives cardinales des Bregs (IL-10, TGF-β1 et IL-35). Nous montrons que les cellules B en latence III de l’EBV peuvent conduire à la mort des cellules T CD4 autologues, ainsi qu’à l’inhibition de la prolifération des lymphocytes T CD4 et CD8, au profit de l’expansion de lymphocytes T régulateurs (Tregs). Nous avons trouvé que cette expansion est médiée par l’axe PD-1/PD-L1. Ces travaux mettent en évidence un nouveau mécanisme de l’EBV concernant le détournement du système immunitaire de l’hôte, augmentant ses capacités oncogéniques. / The Epstein-Barr virus (EBV) is the first transforming virus discovered in humans. It infects more than 90% of the global adult population, persisting in an episomal form in the memory B-cell compartment throughout the life of the host. During primo-infection and during phases of viral reactivation, immortalized B-cells are in latency III, also called the proliferation program, in which the full range of latency proteins are expressed. In immunocompetent subjects, a balance between virus and host is established, and most infected B-cells are eliminated by the host’s immune system, mainly by cytotoxic T lymphocytes. Deficit of the immune system may lead to lymphomagenesis, such as post-transplantation lymphoproliferative disorders (PTLDs), non-Hodgkin’s (NHL) or Hodgkin’s lymphomas (HL). Previous studies in the lab revealed that the immuno-inhibitor PD-L1/B7-H1/CD274 was overexpressed on the surface of EBV latency III B-cells. Interleukin-10 (IL-10was also secreted by these cells. These features are shared with regulatory B-cells (Bregs). Our objective was to examine the immunomodulatory features of EBV latency III B-cells, in the frame of Bregproperties. We found that EBV latency III B-cells possessed the antigenic determinants common to immature Bregs (CD24High CD38High PD-L1High), associated with overexpression of Breg immunosuppressive cytokines (IL-10, TGF-β1 and IL-35). EBV latency III B-cells led to death of autologous CD4 T-cells, as well as inhibition of CD4 and CD8 T-lymphocyte proliferation, favoring regulatory T-cell (Treg) expansion. We found that this expansion was mediated by the PD-1/PD-L1 axis. This study highlights a new mechanism of EBV for t diversion of the host immune system thereby increasing its oncogenic properties.