Genomic dissection of tumor heterogeneity and progression

肿瘤异质性和进展的基因组解剖

• 批准号: 10702643
• 负责人: John Shern
• 金额: $ 76.2万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702643
• 关键词: Bar Codes; Benign; Biological Assay; Biological Models; Biopsy; Biopsy Specimen; Blood specimen; Bromodomain; Cell Communication; Cells; Cellular Assay; Childhood Solid Neoplasm; Chromosome 8; Clinical; Clinical Trials; Complex; Coupled; DNA sequencing; Data; Data Set; Disease; Disease Progression; Dissection; EWS-FLI1 fusion protein; Early Diagnosis; Early treatment; Emerging Technologies; Enrollment; Epigenetic Process; Evolution; Ewings sarcoma; FDA approved; FOXO1A gene; Foundations; Fusion Oncogene Proteins; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genetic Engineering; Genetic Heterogeneity; Genetic Transcription; Genomics; Goals; Heterogeneity; Histologic; Human; Image; Immune; In Vitro; Knock-out; MEKs; Malignant - descriptor; Measures; Methods; Methylation; Modeling; Myeloid Cells; NF1 gene; Nerve; Neurofibrosarcoma; Operative Surgical Procedures; PAX3 gene; PRC1 Protein; Patients; Pediatric Neoplasm; Plexiform Neurofibroma; Population; Pre-Clinical Model; Procedures; Property; Publications; Publishing; Recurrence; Recurrent disease; Refractory Disease; Research Personnel; Resistance; Rhabdomyosarcoma; Sampling; Sensitivity and Specificity; Sirolimus; Solid Neoplasm; Specimen; Staging; System; Technology; Time; Topoisomerase Inhibitors; Universities; Washington; Work; base; blood fractionation; cancer stem cell; cell free DNA; cell type; deep sequencing; design; evidence base; experimental study; genome sequencing; improved; in vivo Model; inhibitor; knockout gene; mTOR Inhibitor; neoplastic cell; neurofibroma; novel; pressure; programs; resistance mechanism; response; single cell sequencing; single-cell RNA sequencing; stem-like cell; therapy resistant; tool; tumor; tumor DNA; tumor heterogeneity; tumor microenvironment; tumor progression; whole genome

Project Summary The first aim of this project is to use single cell sequencing to understand the complexities of cell types and cell to cell heterogeneity that is present within pediatric solid tumors. In this work, we are focusing on generating comprehensive gene expression profiling of the cells present within tumors that occur in patients with NF1. To date we have collected and analyzed surgical specimens from Plexiform Neurofibromas (PN), Atypical Neurofibromas (AN) and Malignant Peripheral Nerve Sheath Tumors (MPNST). Over the past two years, we have generated high gene coverage sequencing on 600,000 single cells from 24 patients with NF1 nerve tumors including histologically validated PN, ANF and MPNST. Our dataset generated to date includes single-cell sequencing of PN and ANF. From these experiments, we capture the landscape of cellular heterogeneity within these tumors. Within PNs we have identified at least 21 unique cell populations including a variety of stromal and immune cell types. This rich dataset details the transcriptional profile of each of these populations and highlights both known and novel cell types. Following the same procedure, we have generated scRNAseq data from 300,000 cells from three MPNST tumors and 10 PDX MPNST models. Preliminary examples of this dataset were published this year in collaborative efforts with investigators at Memorial Sloan Kettering and Washington University. These works discovered the recurrence and importance of chromosome 8 amplification in MPNST and an interesting cellular population with cancer stem cell like properties. Additionally, we have made progress integrating the human findings with genetically engineered model systems of NF1 based nerve tumors. This initial dataset describing the multitude of cell-to-cell interactions within these tumors is accepted for publication. We also have actively been working to discover novel ways to arrest the malignant progress in these tumors. A current effort is to develop an in vitro system to understand the polarization of myeloid cell populations in the tumor microenvironment. If successful, this work could deliver specific vulnerabilities that could be used in combination with the FDA approved MEK inhibitors to further improve tumor shrinkage in patients with plexiform neurofibroma. Given the difficulty in obtaining multiple sequential tumor biopsy specimens from patients with solid tumors, we have undertaken a project to assay cell free DNA to assess disease status in NF1 patients. In this work, we have developed an assay that marries low pass whole genome sequencing with NF1 specific targeted capture deep sequencing of selected genes. This work has culminated in publication of our "classifier" which uses tumor fraction from blood specimens to provide a clinical tool that could be used to differentiate between MPNST and the benign Plexiform Neurofibroma. Currently we are evaluating our assays value using samples collected on the actively enrolling clinical trial SARC031 (NCT03433183) "MEK Inhibitor Selumetinib (AZD6244) in Combination with the mTOR Inhibitor Sirolimus for Patients With MPNST". Another application of cell free DNA technology is correlating the changes observed in the circulating tumor DNA with the changes observed on re-staging imaging as a measure of response of a tumor to therapy. These efforts are currently underway. Secondary efforts to pair the circulating tumor DNA with an on-treatment tumor biopsy to observe correlation and description of the global genomic changes in the circulating tumor DNA to discover mechanisms of tumor evolution are being explored. Finally, we have continued efforts to improve the sensitivity and specificity of the cell free DNA assay. Current efforts include deeper profiling of fragment size, deeper sequencing through capture based or whole genome methods, and alternative sequencing strategies such as methylation profiling. The second aim of this work is to develop and employ novel barcoding strategies married with single cell sequencing that can be used in preclinical model systems to model tumor cell resistance and survival. Single cell sequencing can dissect the gene expression profile of thousands of cells in parallel but is limited in its ability to track populations of cells under a selective pressure. Within the current year we have completed experiments that highlighted the need to add a feature to our experimental system which enabled tracking a particular cell over time and as it replicated. Current experiments are employing the single cell barcoding strategy as well as incorporation of CRISPr gene knockouts to the experimental workflow. We now have completed a screen targeting knockout of multiple epigenetic complexes including SWI/SNF and PRC1 in MPNST cells. These experiments have delivered novel vulnerabilities of the MPNST cell. Ongoing work is designed to validate these observations and describe the mechanisms underlying these observations.

项目摘要 该项目的第一个目标是使用单细胞测序来了解儿科实体瘤中存在的细胞类型的复杂性和细胞间的异质性。在这项工作中，我们的重点是生成 NF1 患者肿瘤内细胞的全面基因表达谱。迄今为止，我们已经收集并分析了丛状神经纤维瘤（PN）、非典型神经纤维瘤（AN）和恶性周围神经鞘瘤（MPNST）的手术标本。在过去的两年里，我们对 24 名 NF1 神经肿瘤患者的 600,000 个单细胞进行了高基因覆盖率测序，包括经过组织学验证的 PN、ANF 和 MPNST。 我们迄今为止生成的数据集包括 PN 和 ANF 的单细胞测序。从这些实验中，我们捕捉到了这些肿瘤内细胞异质性的情况。在 PN 中，我们已经鉴定出至少 21 个独特的细胞群，包括各种基质细胞和免疫细胞类型。这个丰富的数据集详细介绍了每个群体的转录概况，并突出显示了已知和新颖的细胞类型。按照相同的程序，我们从来自三个 MPNST 肿瘤和 10 个 PDX MPNST 模型的 300,000 个细胞生成了 scRNAseq 数据。该数据集的初步示例于今年与纪念斯隆凯特琳大学和华盛顿大学的研究人员合作发布。这些工作发现了 MPNST 中 8 号染色体扩增的复发和重要性，以及具有癌症干细胞样特性的有趣细胞群。此外，我们在将人类发现与基于 NF1 的神经肿瘤的基因工程模型系统相结合方面取得了进展。这个描述这些肿瘤内多种细胞间相互作用的初始数据集已被接受发表。我们还一直积极致力于发现阻止这些肿瘤恶性进展的新方法。目前的努力是开发一种体外系统来了解肿瘤微环境中骨髓细胞群的极化。如果成功，这项工作可以提供特定的漏洞，可以与 FDA 批准的 MEK 抑制剂联合使用，以进一步改善丛状神经纤维瘤患者的肿瘤缩小。鉴于从实体瘤患者身上获取多个连续肿瘤活检标本的困难，我们开展了一个项目，通过检测细胞游离 DNA 来评估 NF1 患者的疾病状态。在这项工作中，我们开发了一种将低通全基因组测序与所选基因的 NF1 特异性靶向捕获深度测序相结合的检测方法。这项工作最终发表了我们的“分类器”，它使用血液样本中的肿瘤部分来提供一种临床工具，可用于区分 MPNST 和良性丛状神经纤维瘤。目前，我们正在使用积极招募的临床试验 SARC031 (NCT03433183)“MEK 抑制剂塞美替尼 (AZD6244) 与 mTOR 抑制剂西罗莫司联合治疗 MPNST 患者”中收集的样本来评估我们的检测价值。无细胞 DNA 技术的另一个应用是将循环肿瘤 DNA 中观察到的变化与重新分期成像中观察到的变化相关联，作为肿瘤对治疗反应的衡量标准。这些努力目前正在进行中。目前正在探索将循环肿瘤 DNA 与治疗中的肿瘤活检配对的二次努力，以观察循环肿瘤 DNA 中整体基因组变化的相关性和描述，从而发现肿瘤进化的机制。最后，我们不断努力提高无细胞 DNA 测定的灵敏度和特异性。 目前的工作包括更深入地分析片段大小、通过基于捕获或全基因组方法进行更深入的测序，以及甲基化分析等替代测序策略。这项工作的第二个目标是开发和采用与单细胞测序相结合的新型条形码策略，可用于临床前模型系统来模拟肿瘤细胞的抵抗和存活。单细胞测序可以并行剖析数千个细胞的基因表达谱，但其在选择压力下追踪细胞群的能力受到限制。在今年内，我们完成了实验，强调需要在我们的实验系统中添加一个功能，该功能能够随着时间的推移和复制而跟踪特定的细胞。目前的实验采用单细胞条形码策略以及将 CRISPr 基因敲除纳入实验工作流程。我们现在已经完成了针对 MPNST 细胞中多个表观遗传复合物（包括 SWI/SNF 和 PRC1）敲除的筛选。这些实验揭示了 MPNST 细胞的新弱点。正在进行的工作旨在验证这些观察结果并描述这些观察结果背后的机制。