Christian Steidl

Mediastinal B-cell lymphomas are lymphoid malignancies that arise in or primarily involve the mediastinum, most prominently primary mediastinal large B-cell lymphoma (PMBCL) and a subset of classical Hodgkin lymphoma (CHL). These entities share overlapping genetic and transcriptional features and are thought to represent a biological continuum originating in the anterior mediastinum. Despite extensive molecular characterization of malignant cells, the role of tissue context and cell–cell interactions in shaping disease phenotype, progression, and relapse remains poorly understood. The overarching goal of this thesis is to define how tissue-specific immune ecosystems influence the biology of mediastinal lymphomas.Using an integrated framework combining imaging mass cytometry (IMC), single-cell RNA sequencing (scRNA-seq), single-cell BCR sequencing and single cell multiomic profiling, this work demonstrates that mediastinal lymphomas are best understood as ecosystem-driven malignancies. In diagnostic CHL, high-dimensional spatial profiling reveals reproducible and genetically linked tumor microenvironment architectures. Genetically defined subgroups of CHL are associated with distinct spatial ecosystem states, indicating that malignant cell genotype is translated into tissue-level immune organization rather than acting independently of the microenvironment.Extending this framework to CHL reveals that relapsed disease represents a biologically distinct ecosystem state rather than simple persistence of diagnostic disease. Single-cell and spatial analyses identify an underappreciated role for non-malignant B cells, demonstrating enrichment of Galectin-9+ naïve B cells in early-relapse CHL and their spatial association with immunosuppressive T-cell populations. These findings highlight immune microenvironment remodeling as a critical feature of relapse biology.Finally, multiomic and spatial characterization of human thymic B cells provides insight into the tissue-restricted biology of PMBCL. This work supports a model in which PMBCL arises from thymus-imprinted B-cell states shaped by local immune regulation rather than classical germinal center differentiation.Together, this thesis establishes a unified ecosystem-based framework for understanding mediastinal lymphomas, emphasizing that malignant evolution, immune regulation, and clinical behavior emerge from coordinated interactions within specific tissue contexts. These findings have implications for biomarker development and for therapeutic strategies that target both malignant cells and the immune ecosystems that sustain them.

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Aggressive B-cell lymphomas encompass a heterogeneous group of neoplasms unified by their rapid clinical course and potential for cure with appropriate therapy. Traditional classification systems, grounded in morphology, often fail to capture the molecular diversity that shapes prognosis and therapeutic response. While molecular profiling has refined classification in diffuse large B-cell lymphoma (DLBCL), other entities remain poorly defined. Among these, high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBCL-DH/TH) and high-grade B-cell lymphoma, not otherwise specified (HGBCL-NOS), represent challenging diagnostic groups with uncertain biological underpinnings. This thesis aimed to define the molecular landscape of HGBCL-DH/TH and HGBCL-NOS to refine their classification and support a biologically informed taxonomy of aggressive B-cell lymphomas. To achieve this, we assembled large study cohorts with detailed clinicopathological data and performed extensive molecular profiling. In HGBCL-DH/TH, tumors with MYC and BCL6 rearrangements exhibited marked heterogeneity, indicating that they do not constitute a single diagnostic group. In contrast, tumors with MYC and BCL2 rearrangements (HGBCL-DH-BCL2) were biologically uniform and exhibited a characteristic gene expression signature. Molecular profiling demonstrated that in HGBCL-DH-BCL2, cooperative genetic alterations converge to dysregulate pathways active in dark zone germinal center B-cells, and that its characteristic gene expression signature closely aligns with transcriptomic profiles of dark zone B-cells. Expression of this signature, termed the dark zone signature (DZsig), extends beyond HGBCL-DH-BCL2 to identify a broader group of lymphomas unified by a dark zone phenotype, providing a biologically informed framework for classification and highlighting potential therapeutic vulnerabilities. Analysis of HGBCL-NOS confirmed that this category is not biologically cohesive but instead comprises tumors with features aligning with established molecular subgroups. The DZsig resolves a substantial fraction of HGBCL-NOS as dark zone lymphomas, while additional cases can be reclassified through genetics-based subtyping of DLBCL, underscoring the limited utility of HGBCL-NOS as a standalone category. Collectively, these findings demonstrate that morphology-based systems alone obscure meaningful biological distinctions. The addition of molecular profiling clarifies the taxonomy of aggressive B-cell lymphomas, enabling refinement of HGBCL-DH/TH and resolution of HGBCL-NOS into molecularly defined subgroups. This framework provides greater diagnostic precision and lays the foundation for developing targeted therapeutic strategies.

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Diffuse large B cell lymphoma (DLBCL) and classic Hodgkin lymphoma (cHL) are two distinct lymphoma entities characterized by vastly different tumour microenvironments. Recurrent mutations in oncogenic signaling pathways, such as NF-κB and JAK-STAT, play key roles in driving the pathogenesis and shaping the microenvironments of these lymphomas. In this thesis, I describe the discovery and functional characterization of somatic loss of the NF-κB regulator TRAF3, and truncating mutations of the cytokine receptor IL4R. Focal deletions of TRAF3 were found in 24/324 DLBCL cases (~7.4%) by high-resolution SNP arrays. CRISPR-mediated knockout of TRAF3 in DLBCL-derived cell lines culminated in constitutive non-canonical (NC) NF-κB pathway activation, rendering these cells sensitive to knockdown or pharmacological inhibition of the central NC NF-κB kinase NIK. TRAF3 ablation further led to exacerbated secretion of the immunosuppressive cytokine IL-10, which impaired induction of Granzyme B and IFNγ of co-cultured CD8+ T cells and restored upon neutralization of IL-10. Additionally, in collaboration, genomic profiling of cHL by deep sequencing of liquid biopsy-derived cell-free DNA identified a novel class of recurrent frameshifting mutations predicted to truncate the IL4R protein in approximately 10% of cHL patients. These mutations clustered around the cytoplasmic tail of the receptor, wherein the immunoreceptor tyrosine-based inhibitor motif (ITIM) resides. HL cells engineered with IL4R truncating mutations exhibited IL-13-dependent upregulation of STAT6 transcription factor phosphorylation and CCL17 chemokine secretion compared to wildtype (WT) controls, which were fully reversible with surface receptor-targeting IL4R neutralizing antibodies. Taken together, our results suggest TRAF3 and IL4R mutations as novel drivers of NC NF-κB and JAK-STAT signaling in DLBCL and cHL, respectively. We further provide proof-of-concept evidence for the targetability of these oncogenic signaling pathways with implications for future therapeutic strategies.

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Non-Hodgkin lymphomas constitute a conglomerate of malignancies that originate from a population of white blood cells termed lymphocytes. Lymphoid cancers are a significant health burden in Canada, with over 9,200 new diagnoses in 2018. Of those newly diagnosed, roughly 40% will die within five years. Studying a subpopulation of aggressive B-cell derived non-Hodgkin lymphomas with both high-throughput sequencing and conventional research techniques, we identified recurrent, somatic aberrations in two genes that are contributors to lymphoma pathogenesis: programmed death ligands 1 (CD274) and 2 (PDCD1LG2). In healthy individuals, programmed death ligands are central to maintaining peripheral tolerance and preventing autoimmune disease. However, we establish that malignant B-cells hijack this axis to suppress the antitumor immune response.Within this dissertation, we report on the frequency of programmed death ligand structural genomic rearrangements in seven different B-cell lymphoma entities and demonstrate the effects of structural genomic rearrangements on gene expression. Additionally, we evaluate ectopic cytokine stimulation, copy number variations, DNA methylation, and micro RNA regulation as additional mechanisms of deregulating programmed death ligand expression. Using novel, capture-based high-throughput techniques, we characterize, at base-pair resolution, the subtypes, cluster locations, and partners of programmed death ligand structural genomic rearrangements. We go on to demonstrate the effects of protein expression and characterize the functional impairment resulting from deregulated programmed death ligand binding in both B and T-cell populations using retroviral cell line models. Finally, in evaluating flanking breakpoint sequences of programmed death ligand structural genomic rearrangements, we propose the molecular mechanisms involved in producing these aberrations. Taken together, our work informs on relevant components of B-cell non-Hodgkin lymphoma pathogenesis and substantiates the effectiveness of molecularly precise therapies targeting the programmed death ligand axis, as they become mainstays of lymphoma treatment.

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Emerging evidence that the interplay between tumour cells and reactive immune cells has profound impact on tumour development, evolution and progression inspired the field of cancer research for the last decade. It has become apparent that the evolutionary pressure exerted by the immune system leads to the evolution of various mechanisms by which tumour cells escape immune surveillance. These often include somatically acquired genetic alterations, resulting in disturbed expression of surface molecules or an altered chemokine/cytokine milieu. B cells play an important role in the adaptive immune response and are potent antigen-presenting cells with high expression of major histocompatibility complexes (MHC) I and II. Multiple studies have reported on defective antigen presentation pathways in malignant lymphomas, however, many of the underlying genetic alterations are largely unexplored.Herein, we applied next generation sequencing techniques and fluorescence in situ hybridization to explore the landscape of genetic alterations in CIITA, the master transcriptional regulator of MHC class II, in various B cell lymphomas and to determine the spectrum of rearrangement partner genes. The functional impact of these mutations on MHC class II expression and the composition of the tumour microenvironment were subsequently evaluated in cell line model systems, and by immunohistochemistry performed on primary lymphoma specimens. Finally, we integrated our findings with patient outcomes to ascertain the clinical impact.We discovered that genomic rearrangements and coding sequence mutations in CIITA are frequent across B cell lymphoma subtypes and can result in diminished MHC class II expression, coinciding with a lower abundance of CD4- and CD8-positive T cells in the tumour microenvironment. We identified that at least some of the genetic alterations are likely a byproduct of AID-mediated somatic hypermutation, as evidenced by the co-occurrence of mutations in the non-coding region of CIITA intron 1. In addition, we described novel translocations involving a broad spectrum of rearrangement partner genes and intra-chromosomal structural variants.In summary, we established CIITA genetic alterations as a frequent immune escape strategy exploited by a variety of malignant B cell lymphomas. These mutations resulted in reduced MHC class II expression and altered microenvironment composition.

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B-cell lymphomas are lymphoid neoplasms derived from mature B lymphocytes at various stages of B-cell development. Advances in sequencing have contributed to decoding the genomic landscapes underlying many subtypes of B-cell lymphomas. However, it remains unclear why some B-cell lymphoma patients suffer from disease progression. A major factor contributing to disease progression is tumour heterogeneity, a consequence of branched evolutionary processes, and microenvironment heterogeneity leading to variation in the composition and properties of non-malignant cells infiltrating and surrounding the cancer. A thorough characterization of these forms of diversity in B-cell lymphomas and their association with disease progression has not been undertaken. As such, the overarching hypothesis of this thesis is that uncharacterized inter-tumour, intra-tumour, and tumour microenvironment heterogeneity impacts disease progression in B-cell lymphomas. In particular, this thesis is focused on studying these types of heterogeneity in three subtypes of B-cell lymphomas and their implications on disease progression. First, I explored inter-tumour heterogeneity in primary specimens of diffuse large B-cell lymphoma patients. I identified novel RCOR1 deletions and their corresponding transcriptional signature in a subset of patients that stratified patients into good and poor outcome following first-line treatment. Secondly, I explored intra-tumour heterogeneity in histologically transformed and early progressed follicular lymphoma patients using serial samples of their primary and transformed/progressed specimens. Through the inference of clonal dynamic patterns, I revealed divergent evolution patterns and identified novel genes underlying these distinct clinical end points. Thirdly, I explored tumour microenvironment (TME) heterogeneity in classical Hodgkin lymphoma relapse patients through serial sampling of primary pretreatment and relapse specimens. I demonstrated how specific TME dynamic patterns can inform on treatment failure. Moreover, I derived a novel, clinically applicable prognostic model (RHL30), based on the TME composition at relapse that predicts response to second-line treatment.Collectively, the work in this thesis constitutes a step forward in our characterization of tumour and microenvironment heterogeneity in B-cell lymphomas and its association with disease progression. The results presented here will aid in the determination of precise therapeutic approaches for individual lymphoma patients.

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The clonal evolution theory of cancer has been recognized for decades and follows principles of Darwinian selection, in which there is selection of the fittest clones in an ecosystem that is fundamentally heterogeneous and undergoes selective pressure. Follicular lymphoma (FL) emerges as a prototypical disease in which to study clonal evolution. It is the most common indolent lymphoma and, although the median overall survival largely surpasses 10 years, patients almost invariably experience progressive disease. Furthermore, a subset of FL patients is at risk of early lymphoma-related death due to rapid progression or transformation to aggressive lymphoma. Yet, the clonal dynamics and the landscape of genomic alterations underlying progression and transformation remain to be uncovered.Herein, we applied whole genome sequencing to a discovery set of transformed, progressed and non-progressed FL cases, re-constructed clonal phylogenies, and interrogated a larger set of transformed FLs and clinical extremes by capture-based targeted sequencing. Moreover, we applied the Lymph2Cx cell-of-origin assay to determine whether molecular subtypes can be defined in transformed follicular lymphoma (TFL) by gene-expression profiling.We discovered that transformation is typically the result of drastic clonal shifts during which TFL-specific clones rapidly outcompete indolent clones. In a subset of cases, these aggressive clones can be found at low levels ( 1% of tumour cells) at diagnosis. In contrast, primary progression generally results from the outgrowth of subclones that are readily detectable at diagnosis, suggesting that the genomic features conferring treatment resistance can be detected prior to initial treatment using low-pass sequencing technology. In addition, we identified discrete gene mutations that are associated with early progression and transformation, and uncovered that a subset of TFLs (16%) have an activated B-cell phenotype and are enriched for mutations in CD79B and BCL10.In summary, we found striking contrast in clonal trajectories between distinct clinical scenarios and described novel associations of gene mutations with transformation and progression. Our findings have translational relevance as they suggest that early progression, and to a lesser extent transformation, can potentially be predicted at diagnosis and thus provide a rationale for novel therapeutic approaches in TFL.

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Classical Hodgkin lymphoma (HL) and primary mediastinal large B cell lymphoma (PMBCL) are related lymphomas sharing pathological, molecular and clinical characteristics. Here we discovered by next-generation sequencing recurrent somatic coding-sequence mutations in the protein tyrosine phosphatase PTPN1 and the cytokine receptor IL4R. Mutations in PTPN1 were found in 6 of 30 (20%) HL cases, in 6 of 9 (67%) HL–derived cell lines, in 17 of 77 (22%) PMBCL cases and in 1 of 3 (33%) PMBCL-derived cell lines, consisting of nonsense, missense and frameshift mutations. We demonstrate that PTPN1 mutations lead to reduced phosphatase activity and increased phosphorylation of JAK-STAT pathway members. Moreover, silencing of PTPN1 by RNA interference in HL cell line KM-H2 resulted in hyperphosphorylation and overexpression of the downstream oncogenes BCL6 and MYC. Mutations in IL4R were found in 18 of 65 (28%) PMBCL cases confirming a ‘hotspot’ missense mutation I242N in exon 8 in 11 of 18 (61%) mutated cases. Ectopic expression of the mutant I242N in HEK 293 cells showed increased activated STAT6-dependent SEAP reporter gene expression without interleukin-4 stimulation. Introduction of the mutant into Hodgkin lymphoma cell line DEV showed cytokine-independent hyperphosphorylation of JAK-STAT pathway members and upregulation of the T cell regulatory chemokine TARC (CCL17) and the B cell activation marker CD23. Our data suggest loss-of-function PTPN1 and gain-of-function IL4R mutations leading to oncogenic JAK-STAT activation as new driver alterations in lymphomagenesis with implications for future treatment strategies.

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