The immune microenvironment in mantle cell lymphoma : Targeted liquid and spatial proteomic analyses

The complex interplay of the tumour and immune cells affects tumour growth, progression, and response to treatment. Restorationof effective immune response forms the basis of onco-immunology, which further enabled the development of immunotherapy. Inthe era of precision medicine, pin-pointing patient biological heterogeneity especially in relation to patient-specific immunemicroenvironment is a necessity for the discovery of novel biomarkers and for development of patient stratification tools for targetedtherapeutics. Mantle cell lymphoma (MCL) is a rare and aggressive subtype of B-cell lymphoma with poor survival and high relapserates. Previous investigations of MCL have largely focused on the tumour itself and explorations of the immune microenvironmenthave been limited. This thesis and the included five papers, investigates multiple aspects of the immune microenvironment withrespect to proteomic analysis performed on tissue and liquid biopsies of diagnostic and relapsed/refractory (R/R) MCL cohorts.Analyses based on liquid biopsies (serum) in particular are relevant for aggressive cases such as in relapse, where invasiveprocedures for extracting tissues is not recommended. Thus, paper I-II probes the possibility of using serum for treatment andoutcome-associated biomarker discovery in R/R MCL, using a targeted affinity-based protein microarray platform quantifyingimmune-regulatory and tumor-secretory proteins in sera. Analysis performed in paper I using pre-treatment samples, identifies 11-plex biomarker signature (RIS – relapsed immune signature) associated with overall survival. Further integration of RIS with mantlecell lymphoma international prognostic index (MIPI) led to the development of MIPIris index for the stratification of R/R MCL intothree risk groups. Moreover, longitudinal analysis can be important in understanding how patient respond to treatment and thiscan further guide therapeutic interventions. Thus, paper II is a follow-up study wherein longitudinal analyses was performed onpaired samples collected at pre-treatment (baseline) and after three months of chemo-immunotherapy (on-treatment). We showhow genetic aberrations can influence systemic profiles and thus integrating genetic information can be crucial for treatmentselection. Furthermore, we observe that the inter-patient heterogeneity associated with absolute values can be circumvented byusing velocity of change to capture general changes over time in groups of patients. Thus, using velocity of change in serumproteins between pre- and on-treatment samples identified response biomarkers associated with minimal residual disease andprogression. While exploratory analysis using high dimensional omics-based data can be important for accelerating discovery,translating such information for clinical utility is a necessity. Thus, in paper III, we show how serum quantification can be usedcomplementary tissue-identified prognostic biomarkers and this can enable faster clinical implementation. Presence of CD163+M2-like macrophages has shown to be associated with poor outcome in MCL tissues. We show that higher expression of sCD163levels in sera quantified using ELISA, is also associated with poor outcome in diagnostic and relapsed MCL. Furthermore, wesuggest a cut-off for sCD163 levels that can be used for clinical utility. Further exploration of the dynamic interplay of tumourimmunemicroenvironment is now possible using spatial resolved omics for tissue-based analysis. Thus, in paper IV and V, weanalyse cell-type specific proteomic data collected from tumour and immune cells using GeoMx™ digital spatial profiler. In paperIV, we show that presence as well as spatial localization of CD163+ macrophage with respect to tumour regions impactsmacrophage phenotypic profiles. Further modulation in the profile of surrounding tumour and T-cells is observed whenmacrophages are present in the vicinity. Based on this analysis, we suggest MAPK pathway as a potential therapeutic target intumours with CD163+ macrophages. Immune composition can be defined not just by the type of cells, but also with respect tofrequency and spatial localization and this is explored in paper V with respect to T-cell subtypes. Thus, in paper V, we optimizeda workflow of multiplexed immunofluorescence image segmentation that allowed us to extract cell metrics for four subtypes ofCD3+ T-cells. Using this data, we show that higher infiltration of T-cells is associated with a positive outcome in MCL. Moreover,by combining image derived metrics to cell specific spatial omics data, we were able to identify immunosuppressivemicroenvironment associated with highly infiltrated tumours and suggests new potential targets of immunotherapy with respect toIDO1, GITR and STING. In conclusion, this thesis explores systemic and tumor-associated immune microenvironment in MCL, fordefining patient heterogeneity, developing methods of patient stratification and for identifying novel and actionable biomarkers

Patient derived lymphoma spheroids (PDLS), un modèle préclinique du lymphome folliculaire : identification de CD39 comme une nouvelle cible thérapeutique potentielle

Le lymphome folliculaire (LF) est le 2ème type de lymphomes non Hodgkiniens (LNH) le plus fréquent, dont le traitement a largement bénéficié de l'introduction de l'anticorps monoclonal anti-CD20, le rituximab en combinaison avec une polychimiothérapie. Malgré ces progrès thérapeutiques considérables, de nombreux patients rechutent et certains patients ne répondent pas au traitement standard. Ainsi, il est primordial d'identifier de nouvelles cibles thérapeutiques à l'aide de modèles pertinents. Ces dernières années ont vu émerger de nouveaux traitements anti-cancéreux "chemo-free". Dans ce contexte, l'immunothérapie a bénéficié d'un puissant développement permettant ainsi, par différentes approches, de stimuler le système immunitaire afin qu'il puisse reconnaître les cellules tumorales et les éradiquer. Le microenvironnement ou la cellule tumorale elle-même sont donc devenus des cibles privilégiées de molécules thérapeutiques. Parmi celles-ci, les molécules ciblant à la fois des récepteurs à l'adénosine ou la génération de son métabolite sont prometteuses. Les modèles 3D représentent un outil d'étude clé dans ce projet car ils reflètent la pathologie en terme : i) d'architecture tridimensionnelle, ii) de profils transcriptomique et protéique et enfin, iii) de réponse aux traitements. Les objectifs spécifiques de ma thèse ont été de: (i) Mettre au point la culture en 3D à partir de lignées cellulaires (MALC, multicellular aggregates of lymphoma cells) et de cellules de patients atteints de LF (PDLS, patient derived lymphoma spheroids), (ii) Caractériser les PDLS sur le plan transcriptomique et phénotypique (composition cellulaire, expression des récepteurs à l'adénosine et ses métabolites, récepteurs d'immune checkpoint...), (iii) Tester l'efficacité d'immunothérapies sur les PDLS et (iv) Identifier de nouvelle(s) cible(s) thérapeutique(s). Ainsi, ce travail a permis d'identifier CD39, une endoectonucléotidase impliquée dans la voie adénosinergique, comme nouvelle cible thérapeutique dans le LF, une pathologie qui reste à l'heure actuelle considérées comme incurable.Follicular lymphoma (FL) is the second most frequent non-Hodgkin's lymphoma (NHL), which benefited largely from anti-CD20 monoclonal antibody (rituximab) introduction in combination with chemotherapy in first-line therapy. Despite this huge therapeutic progress, numerous patients relapse and some are refractory to first-line treatments. Thus, it is essential to identify new therapeutic targets with relevant models. Lately, new anti-cancer treatment emerged called "chemo-free". In this context, immunotherapy has benefited from a powerful development allowing, by different approaches, to stimulate the immune system in order to recognize tumoral cells and eradicate them. Tumor microenvironment or even tumor cells themselves have become privileged targets for those therapeutic molecules. Among them, molecules targeting adenosine receptors or its metabolites generation are promising. 3D models represent a key element in this project as they reflect the pathology in term of: (i) spatial architecture, (ii) transcriptomic and protein profiles, and (iii) treatment responses. The specific objectives of my PhD were to: (i) Establish 3D cultures from FL cell lines (MALC, multicellular aggregates of lymphoma cells) and FL patient samples (PDLS, patient derived lymphoma spheroid), (ii) Characterize the transcriptomic and phenotypic profiles of PDLS (immune cell population composition, adenosinergic pathway markers expression, immune checkpoint receptors...), (iii) Test efficacy of immunotherapies on PDLS and (iv) Identify new therapeutic targets. Thus, this work allowed the identification of CD39, an endoectonucleotidase implicated in adenosine generation, as a new therapeutic target in FL, a pathology that still remains incurable

Development of Multigene Expression Signature Maps at the Protein Level from Digitized Immunohistochemistry Slides

Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions

The evolving landscape of predictive biomarkers in immuno-oncology with a focus on spatial technologies.

Immunotherapies have shown long-lasting and unparalleled responses for cancer patients compared to conventional therapy. However, they seem to only be effective in a subset of patients. Therefore, it has become evident that a greater understanding of the tumor microenvironment (TME) is required to understand the nuances which may be at play for a favorable outcome to therapy. The immune contexture of the TME is an important factor in dictating how well a tumor may respond to immune checkpoint inhibitors. While traditional immunohistochemistry techniques allow for the profiling of cells in the tumor, this is often lost when tumors are analysed using bulk tissue genomic approaches. Moreover, the actual cellular proportions, cellular heterogeneity and deeper spatial distribution are lacking in characterisation. Advances in tissue interrogation technologies have given rise to spatially resolved characterisation of the TME. This review aims to provide an overview of the current methodologies that are used to profile the TME, which may provide insights into the immunopathology associated with a favorable outcome to immunotherapy

Radiomics in [¹⁸F]FDG PET/CT:A leap in the dark?

Positron emission tomography (PET) imaging with the non-metabolisable glucose analogue 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG), combined with low dose computed tomography (CT) for anatomical reference, is an important tool to detect and stage cancer or active inflammations. Visual interpretation of PET/CT images consists of (qualitative) assessment of radiotracer uptake in different tissues and their density. Furthermore, the location, size, shape, and relation with surrounding tissues of these lesions provide important clues on their nature. Yet, medical images contain much more information about tissue biology hidden in the myriad of voxels of both lesions and healthy tissue than can be assessed visually. Quantification of radiotracer uptake heterogeneity and other tissue characteristics is studied in the field of radiomics. Radiomics is a form of medical image processing that aims to find stable and clinically relevant image-derived biomarkers for lesion characterisation, prognostic stratification, and response prediction, thereby contributing to precision medicine. Radiomics consists of the conversion of (parts of) medical images into a high-dimensional set of quantitative features and the subsequent mining of this dataset for potential information useful for the quantification or monitoring of tumour or disease characteristics in clinical practice. This thesis contributed to a deeper understanding of the methodological aspects of handcrafted radiomics in [18F]FDG PET/CT, specifically in small datasets. However, most radiomic papers present proof-of-concept studies and clinical implementation is still far away. At some point in the future, radiomic biomarkers may be used in clinical practice, but at the moment we should acknowledge the limitations of the field and try to overcome these. Only then, we will be able to cross the translational gap towards clinical readiness. Future research should focus on standardisation of feature selection, model building, and ideally a tool that implements these aspects. In such a way, radiomics may redeem the promise of bringing forth imaging biomarkers that contribute to precision medicine.<br/

The function and origin of the CD4+ T cell in the classical Hodgkin lymphoma microenvironment

PhDClassical Hodgkin lymphoma (CHL) is a germinal centre B cell malignancy where the bulk of the tumour comprises a non-clonal immune infiltrate enriched for CD4+ T cells. The role of these cells in the pathophysiology of CHL is poorly understood. Biomarkers predictive of clinical outcome in CHL are limited. This thesis examines microenvironment biomarkers with the goal of identifying the 10-20% of patients who are not cured by conventional therapy, and also investigates the function of the CD4+ T cell in CHL. The prognostic power of FOXP3, a marker of regulatory T cells, CD68, a macrophage marker and CD20, a B cell marker, is validated in a new patient cohort and for the first time CD68 and FOXP3 are combined in a statistically robust scoring system. The data presented challenge the assumption that the microenvironment is Th2-polarised or senescent and demonstrates relative over-expression of T-BET, a Th1 marker and under-expression of PD1, a marker of senescence/exhaustion, with little evidence for Th2 marker expression. A cytokine-enriched in vitro culture system was developed demonstrating superior proliferation and longevity of CHL-derived T cells compared to non-malignant tissue-derived controls. These cells sustain expression of markers associated with proliferation and longevity (e.g. CD27, CD28) and remain functional (express cytokines) for many weeks. A panel of CD4+ T cell-specific markers was determined capable of differentiating CHL-derived from non-malignant or non-Hodgkin lymphoma-derived CD4+ T cells, in which markers of central memory (CD62L and CCR7) and early activation (CD69) are over-represented and markers of senescence (CD57 and PD1) are under-represented. Cytokine profiles were found to resemble Th1 (expression of IL2, IFN- and TNF expression) rather than Th2 (IL4, IL13, IL21, IL10 and IL6) responses. The data presented confirm a new prognostic biomarker signature and show a Th1 rather than Th2-dominated microenvironment enriched for cytokine-secreting functional effector CD4+ T cells and long-lived, proliferative cells resembling central memory cells rather than hypoproliferative, anergic, non-functional T cells

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer\u27s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients