Genomic characterization and development of therapies for pediatric sarcoma

儿科肉瘤的基因组表征和疗法开发

• 批准号: 10486936
• 负责人: John Shern
• 金额: $ 76.02万
• 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/10486936
• 关键词: ATAC-seq; Adolescent; Adolescent and Young Adult; Alveolar; American Society of Clinical Oncology; Benign; Biochemical; Biological; Biological Assay; Biological Models; CLIA certified; CRISPR/Cas technology; CTNNB1 gene; Categories; Cell Cycle; Cells; Chemotherapy and/or radiation; Chemotherapy-Oncologic Procedure; Child; Childhood; Childhood Soft Tissue Sarcoma; Clinical; Clinical Trials; Collaborations; Complex; Consensus; Data; Data Set; Development; Diagnosis; Disease; Enrollment; Epigenetic Process; Event; Ewings sarcoma; FBXW7 gene; FGFR4 gene; FOXO1A gene; Frequencies; Gene Dosage; Genes; Genetic; Genetic Transcription; Genomics; Goals; Histologic; Informatics; KRAS2 gene; Knowledge; Lesion; Localized Disease; Malignant Childhood Neoplasm; Malignant Neoplasms; Molecular; Molecular Biology; Molecular Genetics; Molecular Profiling; Morbidity - disease rate; Mutation; Neurofibromatosis 1; Neurofibrosarcoma; Operative Surgical Procedures; Outcome; PIK3CA gene; Pathway Analysis; Pathway interactions; Patients; Pediatric Oncology; Pediatric Oncology Group; Plexiform Neurofibroma; Polycomb; Population; Pre-Clinical Model; Protocols documentation; Publishing; RAS genes; RNA; Recurrence; Refractory Disease; Relapse; Reporting; Resolution; Rhabdomyosarcoma; Risk; SNP array; Sampling; Signal Transduction; Soft tissue sarcoma; Survival Rate; TP53 gene; Techniques; Toxic effect; Transcription Repressor; Treatment Protocols; United Kingdom; United States; Visualization; WNT Signaling Pathway; Work; aggressive therapy; base; bioinformatics pipeline; childhood sarcoma; clinically relevant; disorder subtype; exome; falls; functional genomics; gene discovery; high risk; high-throughput drug screening; improved; in vitro Model; interest; meetings; member; mortality; multimodality; neoplastic cell; novel therapeutics; patient population; patient stratification; patient subsets; prognostic; prognostic significance; prospective; response; risk stratification; sarcoma; therapeutic target; therapy development; transcription factor; transcriptome sequencing; transcriptomics; tumor; tumorigenesis; user-friendly; whole genome

Project Summary 1. Efforts in Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is a myogenic cancer that is the most common soft tissue sarcoma of childhood. With the development of multimodal chemotherapy regimens, relapse-free survival rates have improved to 70-80% in patients with localized disease, albeit with significant toxicity. Unfortunately, despite aggressive therapy, the 5-year survival rate for patients with metastatic disease remains only 30%. Therapy assignment is currently based on clinicopathologic features and using these criteria, three distinct subgroups of patients can be identified (low, intermediate and high risk). However, many patients fall into the intermediate risk category (which accounts for about 50% of all patients) and have a heterogeneous clinical outcome. This suggests that some of these children could be treated with less aggressive therapy or alternatively should be considered to have more aggressive disease. In an effort to further characterize the genetic events underlying this tumor type, our group in collaboration with the Children's Oncology Group (COG) performed a large sequencing effort using a combination of whole-genome, whole-exome and whole-transcriptome sequencing along with high resolution SNP arrays to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Our findings describe a heterogenous group of genetic events appears to drive RMS most notably the PAX 3/7-FOXO1 fusion in the alveolar subtype and mutation of multiple RAS pathway genes in fusion negative tumors including recurrent genetic lesions in 10 cancer consensus genes (NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1). While the majority of these mutations appear to be mutually exclusive, a subset of tumors appears to have coexisting lesions within the same tumor; perhaps indicating a biologically relevant progression in these tumors. Unfortunately, much of the clinical annotation for these cases was incomplete, severely limiting our ability to derive prognostic information from this data set. To overcome this, we performed a more focused retrospective analysis is needed to determine the prognostic significance of the discovered mutations. In collaboration with the COG and the Shipley group in the United Kingdom, we sequenced 675 clinically annotated rhabdomyosarcoma cases using a clinically relevant capture-based sequencing assay. We have validated that our assay can reliably detect mutations even at low frequency and that we can accurately detect gene copy number changes. In addition, we developed a bioinformatics pipeline and visualization portal that enables a user-friendly interface with the generated data. Clinical outcome correlation analysis was performed in concert with the COG and we have demonstrated the clinical impact of the presence or absence of each genetic lesion. This year we published the initial results of this work (Shern et al 2021) and report that mutations of MYOD1 and TP53 were found to be associated with a worse clinical outcome in fusion negative rhabdomyosarcoma. These results were presented at the American Society of Clinical Oncology Annual meeting as well as the Childrens Oncology Group annual meeting. Current efforts are focused on using these results to enlighten prospective clinical trials. This includes implementation of a CLIA certified assay and informatics pipeline to centrally profile all rhabdomyosarcoma patients enrolled on Children's Oncology Group trials. This effort is being coordinated with the molecular profiling protocol associated with the Childhood Cancer Data Initiative (CCDI) and the COG Project:EveryChild protocol. In addition to these efforts, this year my group continued to generate molecular data associated with patient tumor samples using retrospective samples collected on intermediate and high-risk clinical trials. A current focus of our efforts is to understand the genomic differences between rhabdomyosarcoma that occurs in the adolescent and young adult populations when compared to younger patients. In this work, we are attempting to understand if there are molecular differences between these groups that would explain the observed survival differences between older and younger patients. In addition, we are working to understand the genomic changes that occur in the tumor cell that underlie metastatic and refractory disease. 2. Efforts in Malignant Peripheral Nerve Sheath Tumor (MPNST) The goal of this work is to provide further understanding of the genetic, epigenetic and transcriptomic mechanisms of the molecular oncogenesis underlying the transformation of a Plexiform Neurofibroma to a Malignant Peripheral Nerve Sheath Tumor (MPNST). MPNSTs are a devastating sarcoma that frequently occurs in patients with Neurofibromatosis Type 1 (NF1). Leveraging the expertise and patient population within the Pediatric Oncology Branch, in this project we are using preclinical model systems to dissect the genetic and epigenetic changes that occur as NF1 tumors transition from benign precursors to the aggressive MPNST. Specifically, we are studying the effects of mutation or deletion in one of the polycomb complex (PRC2) members SUZ12 or EED as the final step in the transformation to MPNST. The PRC2 complex is a major transcriptional repressor within the cell and recurrent alterations of members of this complex have been discovered in genomic sequencing studies from patient samples. Within the current fiscal year, we used inducible in vitro model systems and performed ChIPseq, RNAseq and ATACseq to nominate genes whose expression is altered upon loss of these genes with the primary objective to identify genes downstream of the PRC2 loss that are potential therapeutic targets. Not surprisingly, there are hundreds of genes whose expression is altered upon reassembly of the PRC2 complex and includes groups of genes whose expression decreases and those whose expression increases. Pathway analysis of this group of genes demonstrated a marked enrichment for genes involved in PI3K signaling, WNT signaling and cell cycle. Interestingly, the transcriptional changes driven by induction of competent PRC2 imperfectly overlapped with gain of the H3K27me3 mark, whereby only a distinct subset of genes appeared to be directly regulated at the RNA level, by the gain of the repressive mark. Analysis of this interesting group of 145 genes discovered a remarkable enrichment of bivalent genes as the primary downstream targets of PRC2 whose expression changes in response to reintroduction of the competent SUZ12. Of interest are several lineage specific transcription factors that appear to be unique vulnerabilities in MPNST cells. We are currently further interrogating these genes for functional relevance to MPNST using biochemical, molecular biology and CRISPR technologies.

项目概述横纹肌肉瘤（Rhabdomyosarcoma， RMS）是一种肌源性癌症，是儿童时期最常见的软组织肉瘤。随着多模式化疗方案的发展，局部疾病患者的无复发生存率已提高到70-80%，尽管有明显的毒性。不幸的是，尽管有积极的治疗，转移性疾病患者的5年生存率仍然只有30%。目前的治疗分配是基于临床病理特征，并使用这些标准，可以确定三个不同的亚组患者（低、中、高风险）。然而，许多患者属于中等风险类别（约占所有患者的50%），并且具有异质性的临床结果。这表明，这些儿童中的一些可以用不那么积极的治疗，或者应该被认为患有更严重的疾病。为了进一步表征这种肿瘤类型的遗传事件，我们的团队与儿童肿瘤组（COG）合作，使用全基因组、全外显子组和全转录组测序以及高分辨率SNP阵列进行了大量测序工作，以表征147对肿瘤/正常的体细胞改变。我们的研究结果描述了一组异质性的遗传事件似乎驱动RMS，最明显的是肺泡亚型中的PAX 3/7-FOXO1融合和融合阴性肿瘤中多个RAS通路基因的突变，包括10种癌症共识基因（NRAS， KRAS， HRAS, PIK3CA, bor, TP53, NF1, FGFR4, FBXW7, CTNNB1）的复发性遗传病变。虽然这些突变中的大多数似乎是相互排斥的，但有一部分肿瘤似乎在同一肿瘤内存在共存的病变；也许表明了这些肿瘤的生物学相关进展。不幸的是，这些病例的许多临床注释是不完整的，严重限制了我们从这些数据集中获得预后信息的能力。为了克服这一点，我们进行了更集中的回顾性分析，以确定发现的突变的预后意义。与COG和英国Shipley小组合作，我们使用临床相关的基于捕获的测序分析对675例临床注释横纹肌肉瘤病例进行了测序。我们已经证实，我们的分析可以可靠地检测突变，即使在低频率，我们可以准确地检测基因拷贝数的变化。此外，我们还开发了一个生物信息学管道和可视化门户，使生成的数据具有用户友好的界面。临床结果相关性分析与COG一起进行，我们已经证明了每种遗传病变存在或不存在的临床影响。今年，我们发表了这项工作的初步结果（Shern et al 2021），并报道MYOD1和TP53突变被发现与融合阴性横纹肌肉瘤较差的临床结果相关。这些结果在美国临床肿瘤学会年会上以及儿童肿瘤小组年会上发表。目前的工作重点是利用这些结果来启发前瞻性临床试验。这包括实施CLIA认证的检测和信息学管道，以集中分析所有参加儿童肿瘤组试验的横纹肌肉瘤患者。这项工作正在与儿童癌症数据倡议（CCDI）和COG项目：每个儿童方案相关的分子分析方案进行协调。除了这些努力之外，今年我的小组继续利用在中高风险临床试验中收集的回顾性样本，生成与患者肿瘤样本相关的分子数据。我们目前的工作重点是了解发生在青少年和年轻成人人群中的横纹肌肉瘤与年轻患者之间的基因组差异。在这项工作中，我们试图了解这些组之间是否存在分子差异，这将解释老年和年轻患者之间观察到的生存差异。此外，我们正在努力了解发生在转移性和难治性疾病基础上的肿瘤细胞的基因组变化。2. 在恶性周围神经鞘肿瘤（MPNST）方面的努力本工作的目的是进一步了解丛状神经纤维瘤向恶性周围神经鞘肿瘤（MPNST）转化的分子癌发生的遗传、表观遗传和转录机制。MPNSTs是一种破坏性肉瘤，常发生在1型神经纤维瘤病（NF1）患者中。利用儿科肿瘤科的专业知识和患者群体，在这个项目中，我们使用临床前模型系统来分析NF1肿瘤从良性前体向侵袭性MPNST转变过程中发生的遗传和表观遗传变化。具体来说，我们正在研究多梳复合体（PRC2）成员之一SUZ12或EED突变或缺失的影响，作为向MPNST转化的最后一步。PRC2复合体是细胞内主要的转录抑制因子，在患者样本的基因组测序研究中发现了该复合体成员的复发性改变。在本财政年度，我们使用了可诱导的体外模型系统，并执行了ChIPseq、RNAseq和ATACseq来指定在这些基因丢失后表达改变的基因，主要目的是鉴定PRC2丢失下游的潜在治疗靶点基因。毫不奇怪，有数百个基因的表达在PRC2复合体重组后发生改变，其中包括表达减少和表达增加的基因群。对这组基因的通路分析表明，与PI3K信号通路、WNT信号通路和细胞周期相关的基因显著富集。有趣的是，由诱导胜任PRC2驱动的转录变化与H3K27me3标记的获得不完全重叠，因此只有一个独特的基因子集似乎在RNA水平上由抑制标记的获得直接调节。对145个基因的分析发现，PRC2的主要下游靶点是显著富集的二价基因，它们的表达在重新引入SUZ12后发生变化。令人感兴趣的是几个谱系特异性转录因子，它们似乎是MPNST细胞中独特的脆弱性。目前，我们正在利用生物化学、分子生物学和CRISPR技术进一步研究这些基因与MPNST的功能相关性。