Genomic dissection of tumor heterogeneity and progression

项目概述本项目的第一个目标是使用单细胞测序来了解儿科实体肿瘤中存在的细胞类型和细胞间异质性的复杂性。在这项工作中，我们专注于生成NF1患者肿瘤内存在的细胞的全面基因表达谱。到目前为止，我们已经收集和分析了丛状神经纤维瘤(PN)、不典型神经纤维瘤(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55名NF1神经肿瘤患者的600,000个单细胞进行了高基因覆盖测序，包括组织学验证的PN、ANF和MPNST。到目前为止，我们生成的数据集包括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中细胞异质性的图景。在三叉神经节内，我们确定了至少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说明了这些群体中每一个的转录特征，并突出了已知和新的细胞类型。按照同样的程序，我们已经从8个MPNST肿瘤和10个PDX MPNST模型的300,000个细胞中产生了scRNAseq数据。这个数据集的初步例子已经在纪念斯隆·凯特林纪念馆和华盛顿大学的研究人员的合作下发表了。这些工作发现了8号染色体扩增在MPNST和一个具有癌症干细胞样特性的有趣细胞群中的复发和重要性。此外，我们还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。这个最初的数据集描述了这些肿瘤内大量的细胞间相互作用，已经接受出版。我们也一直在积极地寻找新的方法来阻止这些肿瘤的恶性进展。目前的一项工作是开发一种体外系统来了解肿瘤微环境中髓系细胞群体的极化。如果成功，这项工作可能会带来特定的脆弱性，可以与FDA批准的MEK抑制剂结合使用，以进一步改善丛状神经纤维瘤患者的肿瘤缩小。考虑到从实体瘤患者获得多个连续的肿瘤活检标本的困难，我们开展了一个项目来检测游离DNA来评估NF1患者的疾病状态。在这项工作中，我们开发了一种结合低通全基因组测序和NF1特定靶向捕获选择基因深度测序的方法。这项工作最终发表了我们的“分类器”，它使用血液标本中的肿瘤部分来提供一种临床工具，可以用来区分MPNST和良性丛状神经纤维瘤。目前，我们正在使用已完成的临床试验SARC031(NCT03433183)“MEK抑制剂Selumetinib(AZD6244)与mTOR抑制剂西罗莫司联合用于MPNST患者”以及正在进行的针对非典型神经纤维瘤患者测试CDK4抑制剂的临床试验(NCT04750928)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中观察到的肿瘤DNA的变化与重新分期成像中观察到的变化相关联，以此来衡量肿瘤对治疗的反应。这些努力目前正在进行中，在过去的一年里，我们与COG软组织肉瘤委员会合作，将无细胞DNA分析纳入积极的临床试验。正在探索将循环肿瘤DNA与正在治疗的肿瘤活检配对，以观察循环肿瘤DNA中整体基因组变化的相关性和描述，以发现肿瘤演变的机制。最后，我们继续努力提高无细胞DNA检测的灵敏度和特异性。目前的努力包括更深入的片段大小测序，通过基于捕获的方法或全基因组方法进行更深层次的测序，以及甲基化测序等替代测序策略。这项工作的第二个目标是使用产生的基因组数据来开发合理选择的组合治疗策略，以克服肿瘤的异质性。在过去的一年里，这项工作得到了极大的扩展，我们在复发的难治性白血病、PAX3-FOXO1驱动的横纹肌肉瘤、EWS-FLI1驱动的尤因肉瘤、MYOD1突变的横纹肌肉瘤和MPNST方面做出了积极的努力。目前，联合策略正在进行体外和体内评估。在接下来的一年里，在活体环境中显示出疗效的成功候选药物将接受潜在临床试验的评估。

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 55 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 eight MPNST tumors and 10 PDX MPNST models. Preliminary examples of this dataset have been published 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 completed clinical trial SARC031 (NCT03433183) "MEK Inhibitor Selumetinib (AZD6244) in Combination with the mTOR Inhibitor Sirolimus for Patients With MPNST" and the ongoing clinical trial testing a CDK4 inhibitor in patients with atypical neurofibroma (NCT04750928). 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 and in the past year we have collaboratively worked with the COG soft tissue sarcoma committee to incorporated cell free DNA assays into active clinical trials. 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 use the generated genomic data to develop rationally selected combination therapeutic strategies to overcome tumor heterogeneity. This work has greatly expanded over the past year and we have active efforts in relapsed refractory leukemia, PAX3-FOXO1 driven rhabdomyosarcoma, EWS-FLI1 driven Ewing Sarcoma, MYOD1 mutant rhabdomyosarcoma, and MPNST. Currently, the combination strategies are being evaluated in vitro and in vivo. In the coming year, successful candidates that show efficacy in the in vivo setting, will be evaluated for potential clinical trials.