3-D spatial approach to discover genomic effectors of immunosuppression during malignant transformation

项目摘要/摘要 IDH突变的低级别胶质瘤(LGG)向侵袭性高级别肿瘤的恶性转化(MT)是一种 具有重大临床意义的事件，最终导致大多数LGG患者死亡。我们发现 IDH突变促进了免疫抑制的微环境，其特征是 LGG中STAT1调节的趋化因子的产生和CD8+T细胞的低浸润在恶性转化中 肿瘤，我们发现了IDH1突变等位基因的意外缺失，这可能会驱动相反的变化 对免疫抑制的微环境，特别是在MT期间。接受治疗诱导的LGG 高突变(HM)是MT的另一条途径，可产生更多高质量的新抗原。总体上说是恶毒的 相对于患者匹配的LGG，我们发现T细胞克隆和 参与细胞毒性T细胞吸引和效应器功能的基因表达增加。基于这些 数据，我们假设IDH突变体LGG的免疫抑制在MT时减少，由基因驱动 主要在恶变过程中获得的改变。为了解决这一假设，我们将 量化MT期间突变的IDH1驱动的免疫抑制的空间和时间变化(目标1)。我们有 设计了一种新的三维(3-D)肿瘤范围的方法，在这种方法中我们将获得10个空间地图 每个肿瘤的样本代表肿瘤的最大解剖结构。整个队列将包括30名恶性肿瘤患者 我们已保存样本的患者的转化和30例非恶性转化复发 匹配的初始IDH1-突变体LgG。我们将使用一种高灵敏度的T细胞谱系分析，通过 飞行时间(CyTOF)、基于RNAseq的去卷积和多重免疫组织化学映射 3-D的免疫格局，并确定突变的IDH1介导的免疫抑制的程度 在MT期间减少。在目标2中，我们将确定在移植过程中获得的基因改变如何影响突变 IDH1介导的免疫抑制。我们将对收集的样本进行深度全外显子组测序 目的1绘制MT过程中肿瘤的三维基因组图谱。我们将测试MT在当地的影响- 相关的基因改变，包括高突变肿瘤中的高质量新抗原，缺失 突变的IDH1等位基因，或其他免疫抑制的遗传事件。了解哪些遗传事件 促进免疫抑制的变化对于选择可以协同作用的靶向治疗至关重要 用免疫疗法来预防或延缓MT。为了开始开发基于T细胞的疗法，我们将捕获 新表位特异性T细胞，优先考虑肿瘤范围内存在的T细胞，并确定 它们针对的新表位/HLA以及相应的T细胞受体(TcR)α和 β链。然后我们将测试克隆的TCR的相对靶标特异性和抗新抗原的活性- 阳性的患者特异性肿瘤细胞。横跨肿瘤大片区域的三维免疫基因组图像 对于设计对整个肿瘤具有活性的个性化治疗是必不可少的。

PROJECT SUMMARY/ABSTRACT Malignant transformation (MT) of IDH-mutant low grade glioma (LGG) to aggressive high grade tumors is an event of major clinical significance, eventually leading to death in the majority of LGG patients. We discovered that mutations in IDH promote an immunosuppressed microenvironment characterized by decreased production of STAT1-regulated chemokines and low CD8+ T cell infiltration in LGG. In malignantly transformed tumors, we identified the unexpected deletion of the IDH1 mutant allele that may drive counteracting changes to the immunosuppressed microenvironment specifically during MT. LGG that undergo treatment-induced hypermutation (HM), another route to MT, produce more high quality neoantigens. Overall in malignantly transformed tumors relative to patient-matched LGG, we found increasing numbers of T cell clones and increasing expression of genes involved in cytotoxic T cell attraction and effector function. Based on these data, we hypothesize that immunosuppression in IDH mutant LGG is reduced upon MT, driven by genetic alterations that are acquired primarily during malignant transformation. To address this hypothesis, we will quantify spatial and temporal changes in mutant IDH1-driven immunosuppression during MT (Aim 1). We have devised a novel 3-dimensional (3-D), tumor-wide approach in which we will acquire 10 spatially mapped samples per tumor representing maximal anatomy of the tumor. The full cohort will include 30 malignantly tranformed and 30 non-malignantly transformed recurrences from patients for which we have banked samples of the matching initial IDH1-mutant LGG. We will use a high-sensitivity T cell repertoire assay, cytometry by Time of Flight (CyTOF), RNAseq based deconvolution, and multiplex immunohistochemistry to map the immunologic landscape in 3-D, and determine the extent to which mutant IDH1-mediated immunosuppression is reduced during MT. In Aim 2, we will determine how genetic alterations acquired during MT affect mutant IDH1-mediated immunosuppression. We will perform deep whole exome sequencing on samples collected in Aim 1 to map the intratumoral genomic landscape in 3-D during MT. We will test for the local influence of MT- associated genetic alterations, including high quality neoantigens in hypermutated tumors, deletion of the mutant IDH1 allele, or other genetic events on immunosuppression. Understanding which genetic events contribute to changes in immunosuppression is critical for selecting targeted therapies that could synergize with immunotherapies to prevent or delay MT. To begin to develop T cell based therapies, we will capture neoepitope-specific T cells, prioritizing those that are present tumor-wide, and determine the neoepitopes/HLAs they target and the amino acid sequences for corresponding T Cell Receptor (TCR) α- and β-chains. We will then test the cloned TCR for relative target specificity and activity against neoantigen- positive patient-specific tumor cells. The 3-D immuno-genomic landscapes across wide swaths of the tumor will be essential to the design of personalized therapies that have activity against the whole tumor.