Dissecting and Modeling Evolution of Inter- and Intra-tumoral Heterogeneity in High-Risk Diffuse Large B-cell Lymphoma

弥漫性大B细胞淋巴瘤是侵袭性淋巴结癌的最常见形式，复发性和难治性弥漫性大B细胞淋巴瘤（rrDLBCL）是致命性疾病，大多数患者死于其疾病。从机制上讲，这是由研究不足的肿瘤间和肿瘤内异质性推动的。我们的研究小组分析了新发DLBCL的肿瘤间异质性（Chapuy et al Nat. Med. 2018），并开始在DLBCL样本的试点系列中探索肿瘤内异质性（ITH），这表明相同患者的淋巴瘤亚群对治疗的反应不同（Roider et al. Nat. Cell Biol. 2020）。值得注意的是，C3和C5 DLBCL亚型是高风险DLBCL亚型，然而，并非所有患者都表现出令人沮丧的结果。在这里，我们的目标是了解这种ITH，并将利用我们以前的发现，并利用现有的原始和rrDLBCL样本的临床注释单细胞悬液的独特资源，成熟的技术和深入的建模专业知识，并提出：1）进行综合多组学分析，以剖析遗传高危C3和C5 DLBCL中遗传和TME因子的细胞内异质性，2）在基因工程细胞系模型和我们建立的暴露于靶向和经典化疗剂后的离体LN模型中，定义和模拟肿瘤内异质性的生物学相关克隆和亚克隆遗传决定因素;以及3）对最简约的可操作标记进行空间分析，并在大型临床注释的淋巴瘤群组中验证它们。重要的是，我们不仅建议采用静态多组学技术，而且还将签名与动态药物反应和功能测量（如凋亡潜力）联系起来。利用我们长期以来的专业知识，我们还将模拟切割的ITH如何导致高风险C3和C5 DLBCL的治疗失败，最终找到靶向治疗失败的新途径和/或开发强大的生物标志物来预测治疗失败。值得注意的是，所有数据将在现有的易于使用但尚未发布的交互式数据门户中共享，使其成为淋巴瘤社区的独特存储库。

Diffuse large B-cell lymphoma is the most common form of aggressive lymph node cancer and relapsed and refractory diffuse large B-cell lymphoma (rrDLBCL) is a fatal disease with the majority of patients dying from their disease. Mechanistically this is fueled by an understudied intertumoral and intratumoral heterogeneity. Our groups dissected intertumor heterogeneity of de novo DLBCL (Chapuy et al Nat. Med. 2018) and started to explore intratumoral heterogeneity (ITH) in a pilot series of DLBCL samples, suggesting that lymphoma subpopulation of the same patients have a differential response to treatments (Roider et al. Nat. Cell Biol. 2020). Notably, C3 and C5 DLBCL subtypes are high-risk DLBCL subtypes in which, however, not all patients exhibit a dismal outcome. Here, we aim to understand this ITH and will capitalize on our previous findings and leverage a readily available unique resource of clinically annotated single-cell suspensions of primary and rrDLBCL samples, established technologies and deep modeling expertise and propose: 1) To perform an integrative multi-omics analysis to dissect the intracellular heterogeneity of genetic and TME factors in genetically high-risk C3 and C5 DLBCLs employing state-of-the-art bulk and single-cell technologies; 2) To define and model biologically relevant clonal and subclonal genetic determinants of intratumoral heterogeneity in genetically engineered cell line models and our established ex vivo LN model following exposure to targeted and classical chemotherapy agents; and, 3) To perform spatial analyses of the most parsimonious actionable signatures and to validate them in large clinically annotated lymphoma cohorts. Importantly, we propose not only to employ static multi-omics technologies, but also link the signatures to dynamic drug responses and functional measurements such as the apoptotic potential. Capitalizing on our longstanding expertise, we will also model how the dissected ITH contributes to treatment failure in high-risk C3 and C5 DLBCLs to eventually find novel avenues to target treatment failure and/or develop robust biomarkers to predict treatment failure. Notably, all data will be shared in an existing easy-to-use and yet unpublished interactive data portal, making it a unique repository for the lymphoma community.