Quantitative Analysis of Carbonic Anhydrase IX Uncovers Hypoxia-Related Functional Differences in Classical Hodgkin Lymphoma Subtypes

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The FYN-TRAF3IP2 gene fusion drives oncogenic NF-κB signaling in peripheral T cell lymphoma

Angioimmunoblastic T cell lymphoma (AITL) and peripheral T cell lymphoma not-otherwise-specified (PTCL, NOS) have poor prognosis and lack actionable targets for directed therapies in most cases. Here we report the identification of FYN-TRAF3IP2 as a novel highly recurrent oncogenic gene fusion in AITL and PTCL, NOS tumors. Mechanistically, FYN-TRAF3IP2 triggers aberrant NF-κB activity by engaging TRAF6 downstream of T cell receptor signaling. Moreover, FYN-TRAF3IP2 expression in hematopoietic progenitors induces NF-κB-driven T cell transformation in mice and cooperates with loss of the Tet2 tumor suppressor in PTCL development. Therapeutically, abrogation of NF-κB signaling in FYN-TRAF3IP2-induced tumors via IκB kinase inhibitors delivers strong anti-lymphoma effects in vitro and in vivo. These results formally demonstrate an oncogenic role for FYN-TRAF3IP2 and NF-κB signaling in the pathogenesis of PTCL

A novel tool for survival analysis in lymphoma patients

Annually, cancer is responsible for 40% of earlier deaths due to non-communicable diseases, and this number increases at an annual rate of around 1.6%. These alarming values make it essential to study this disease at a global level, to help better the lives of all the affected patients and disseminate prevention when possible. With the advance in technology and thanks to the influx of patients with digitalised records that suffer from this disease, there is a greater capability to elaborate a study about the possible causes and consequences drawn from the patient’s data. Furthermore, the ability to better the patient’s quality of life by analysing their data and sensitising them is fundamental in the fight against cancer. The dissertation focuses on developing a computational tool that enables tha ability to obtain simple statistics, thanks to classical techniques of survival analysis as well as the analysis of lymphoma cancer, both Hodgkin and non-Hodgkin lymphomas that constitute nearly 48% of blood cancers. To determine the factors that influence the study of the received patients’ database, a preprocessing is done where the descriptive statistics are obtained using the patients’ database information. After that, Kaplan-Meier estimator curves are elaborated to determine the relationship between the studied phenomenon and the different variables present in the database. After taking brief conclusions from the obtained variables and subsequent descriptive analysis, an analysis using the Kaplan- Meier estimator is done. The integration of the achieved results is implemented in a tool that constitutes CLARIFY 1’s project dashboard. This dissertation was created in conjunction with the CLARIFY European project, led by the oncology medical team of University Hospital Puerta Hierro de Majadahonda.Anualmente, o cancro é responsável por 40% das mortes precoces devido a doenças não transmissíveis, e este valor aumenta anualmente cerca de 1.6%. Estes valores alarman- tes fazem o estudo desta doença um foco fundamental a nível global de modo a melhorar a vida de todos os pacientes e disseminar prevenção a quando possibilidade do mesmo. Com o avançar da tecnologia e graças a um influxo de registos digitalizados sobre pacientes que sofrem este tipo de doença, existe uma maior capacidade de elaborar um estudo sobre as possíveis causas e consequências retiradas a partir dos dados de pacientes que passaram por isso. Para além disso, a capacidade de melhorar a qualidade de vida dos pacientes através da análise dos seus dados e da sensibilização dos mesmos é fundamental para uma constante luta contra o cancro. Esta dissertação foca-se no desenvolvimento de uma ferramenta computacional que permite aceder de forma simples, a estimativas obtidas a partir de técnicas clássicas de análise de sobrevivência como exemplo de aplicação, foca-se ainda na análise do cancro linfoma tanto Hodgkin como não-Hodgkin, que abrange cerca de 48% dos cancros de sangue. Com o objetivo de averiguar os fatores de risco que influenciam a sobrevivência dos pacientes da base de dados em estudo, é efetuado um pré-processamento dos dados, onde são obtidas estatísticas descritivas da base de dados de pacientes e produzidas estatísticas das curvas de sobrevivência com recurso ao estimador de Kaplan-Meier de modo a determinar a relevância das variáveis analisadas da base de dados em relação ao acontecimento analisado. A integração dos resultados obtidos através do estimador Kaplan-Meier será integrada numa ferramenta que por sua vez fará parte do Dashboard do projeto do CLARIFY 2. Esta dissertação foi criada em conjunto com o projeto europeu, Clarify, liderado pela equipa médica de oncologia do Hospital Universitário Puerta Hierro de Majadahond

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

Macrophage and T helper dynamics in Classical Hodgkin Lymphoma and their role in PD1 checkpoint blockade

Classical Hodgkin Lymphoma (CHL) is a germinal centre B cell lymphoma characterised by rare lymphoma cells within a diverse inflammatory infiltrate. It is unusual amongst haematological malignancies in that it responds well to PD1 checkpoint blockade. However, evidence for T cell exhaustion is limited which is surprising given that PD1 checkpoint inhibitors are thought to act via the reversal of exhaustion. This thesis examines the mechanisms by which CHL cells recruit and induce a tolerant myeloid and T cell environment, focussing on the role of the PD1-PDL1 axis and the implications of this for the action of checkpoint inhibitors. The recruitment and induction of a PDL1 positive macrophage phenotype is modelled in vitro and in primary patient samples, confirming the induction of PDL1 by CHL cells. However, no phenotypic or functional evidence of T cell exhaustion is detected above that seen in reactive tissues and no relationship is seen between markers of exhaustion and markers that predict PD1 inhibitor response challenging the current narrative that PD1 inhibitors in CHL work via the reversal of exhaustion. Instead, spatial and in vitro modelling identify T helper differentiation as a putative mechanism for PD1 inhibitor action. They highlight CHL major histocompatibility complex II expression as playing a central role in the induction of a TH1 regulatory cell rich environment but also as a vulnerability for CHL cells once treatment is initiated. As part of this thesis we focus upon multi-parameter imaging analysis. Multi-parameter imaging enables deep phenotyping whilst retaining spatial relationships between cells. However, whilst advances in phenotyping approaches have been made there remains a need for better tools for spatial analysis. We describe the novel application of spatial point pattern modelling to this task and demonstrate the utility and flexibility of this method in deconstructing spatial relationships and interactions

Contribution of tumour cell signalling and the microenvironment to the pathogenesis of EBV-associated B cell lymphoma and

In this thesis I have explored different components of the pathogenesis of several related EBV associated cancers. In the first part of the thesis I focus on the microenvironment of two of these cancers, nasopharyngeal carcinoma (NPC) and diffuse large B cell lymphoma (DLBCL). Our group has developed a therapeutic vaccine against EBV which has already been shown to be safe in patients with NPC. Therefore, in the first results chapter (chapter 3), I present a description of the phenotyping of expression of the immune microenvironment including immune checkpoint (ICP) genes and MHC class I and class II genes in NPC tissues. I showed for the first time in NPC tissue samples, two types of PD-L1 expressing tumours: diffuse and marginal. In re-analysis of published data, I found co-expression of immune checkpoint genes and their receptors in EBV positive NPC samples; information which is likely to inform the design of combination immunotherapy in NPC patients. I have shown in my re-analysis of EBV positive NPC that PD-L1 is not up-regulated by LMP-1. In chapter 4, I show the results of studies of the expression of collagen and collagen receptors in DLBCL in which I have identified the over-expression of a potentially novel immune checkpoint receptor, LAIR-1, on the macrophages infiltrating this tumour. In the second part of the thesis I switch my line of inquiry to the tumour cells of EBV-associated cancers, this time focussing on Hodgkin lymphoma (HL), another EBV-associated lymphoma. During the course of the work presented in this chapter I was able to define a role for aberrant sphingosine-1-phosphate (S1P) signalling in driving PI3-K activation mediated through up-regulation of S1PR1 and downregulation of S1PR2 receptors in HL. I also showed that in turn, PI3-K signalling increases the expression of potentially oncogenic downstream transcription factors, such as BATF3 which I have shown to be overexpressed in HL. These data suggest that antagonists of S1P could be considered for the treatment of patients with HL

Classification of Lymphomas and Hematological Neoplasia in the Era of Genomic Research

This Special Issue contains review articles focusing on several lymphoma entities included in the current WHO classification. The aim of the book is to guide the readers in understanding the evolution of lymphoma classification. The clinicopathological entities described in this issue have been analyzed through the molecular mechanisms involved in their pathogenesis

A Graph-Based Digital Pathology Approach To Describe Lymphocyte Clustering Patterns After Renal Transplantation

Introduction/ Background Renal transplantation (rTx) induces an adaptive immune response against foreign donor antigens mediated by lymphocytes of the recipient. Local accumulation of B- and T-cells is an important component of this response enabling and controlling immune cell interactions [1]. Combining digital microscopic images with network analysis [2][3] opens new perspectives to study the spa- tial dimension of lymphocyte clustering and to model their potential interactions.   Aims The aim of this study is to characterize the range of B- and T-lymphocytic infiltrates below the threshold of rejection defined by theBanffclassification [4][5] and to propose a mathematical description of immune cell clustering for use in systems medicine approaches.   Methods We established a workflow to comprehensively characterize lymphocyte clusters and compare their morphological features with organized structures such as secondary or tertiary lymphoid organs (TLO/SLO) [6]. 51 renal protocol and indication biopsies from 13 patients without evidence for severe rejection over 10 years were stained by CD3/CD20 duplex immunohisto- chemistry. Whole slide images (WSIs) were acquired to automatically detect biologically relevant regions of in- terest (ROIs) by means of density maps for lymphocytes (image analysis workflow illustrated in Fig. 1a). They are generated from single nuclei identification using an au- to-adaptive random forest pixelwise classifier (“nucleus container” module [7],Definiens,Germany). We imple- mented a graph-based tool in Java using individual cell coordinates to identify cell compartments (Fig. 1b) and applied it to each selected ROI. For this, a neighborhood graph is built by Delaunay triangulation and Euclidean distances. This analysis allows describing their specific clustering behavior based on features as described in [8]. The convex hull of the neighborhood graph allows a visualization of B- and T-cell compartments.   Results We identified B-cell rich compartments in about 55% of 150 ROIs in kidney tissue after successful transplantation (examples in Fig. 2). The B-cell compartments in rTx tended towards smaller overall size with on average about 90 cells in a B-cell cluster compared to more than 600 B-cells observed in mature TLOs and SLOs and they showed less prominent spatial organization (average degree on average 3.92 instead of 4.97; degree shows generally Poisson distribution as illustrated in Fig. 3A). Further, the graph analysis confirmed lower B-cell density (Fig. 3B displays the exponential character of the spatial B-cell distribution in a selected ROI), a different ratio between T- and B-cell compartments, and more frequent overlap between both regions than in mature lymphoid structures.   We conclude that the graph-based approach is feasible to distinguish relevant immune cell patterns in rTx and provides a useful mathematical description of neighborhood relationships between immune cells and their spatial organization. The workflow has the potential to improve throughput and robustness of immune cell evaluation for use in translational science

GPNMB: exploring a novel target for therapy in Hodgkin lymphoma

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a transmembrane protein highly expressed in multiple tumours, including classical Hodgkin lymphoma (cHL); a tumour in which the malignant Hodgkin and Reed-Sternberg (HRS) cells rely on the tumour microenvironment (TME) to survive and evade detection by the host immune system. In this study, I explore the contribution of GPNMB to immune evasion of cHL as a novel therapeutic target. I have shown that GPNMB is highly expressed in tumour-associated macrophages (TAM) in cHL tissues but expression is highly variable. In vitro differentiation of macrophages (from CD14$^+$ monocytes) was shown to be associated with an increase in GPNMB expression, including the generation of a soluble form of GPNMB (sGPNMB). sGPNMB partially inhibited T-cell activation and T-cell recognition of Epstein-Barr virus (EBV)-specific epitopes in cHL cell lines. Preliminary experiments indicated that the inhibition of T cell activation by GPNMB could be overcome with GPNMB neutralising antibodies. This work provides evidence in support of the hypothesis that GPNMB can mediate an immune checkpoint that inhibits anti-tumour cytotoxic T-lymphocytes (CTL). It provides a basis for further investigations designed to explore the therapeutic potential of targeting GPNMB in cHL