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: 10014787
• 负责人: John Shern
• 金额: $ 80.14万
• 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/10014787
• 关键词: ATAC-seq; Adolescent; Alveolar; American Society of Clinical Oncology; Benign; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Models; CLIA certified; CRISPR/Cas technology; CTNNB1 gene; Categories; Cells; Chemotherapy-Oncologic Procedure; Child; Childhood; Childhood Soft Tissue Sarcoma; Clinical; Clinical Trials; Collaborations; Complex; Consensus; Data; Data Set; Development; Diagnosis; Disease; Drug Screening; Embryonal Rhabdomyosarcoma; Enrollment; Epigenetic Process; Event; Evolution; Ewings sarcoma; FBXW7 gene; FGFR4 gene; FOXO1A gene; Frequencies; Gene Dosage; Genes; Genetic; Genomics; Goals; Histologic; Histology; Imagery; Informatics; KRAS2 gene; Knowledge; Lesion; Localized Disease; Malignant Neoplasms; Molecular Biology; Molecular Genetics; Morbidity - disease rate; Mutation; NF1 gene; Neurofibrosarcoma; Operative Surgical Procedures; Outcome; PIK3CA gene; Pathology; Pathway interactions; Patients; Pediatric Oncology; Pediatric Oncology Group; Pilot Projects; Polycomb; Pre-Clinical Model; Prognostic Marker; RAS genes; Radiation; Recurrence; Relapse; Reproducibility; Resolution; Rhabdomyosarcoma; Risk; Risk stratification; SNP array; Sampling; Soft tissue sarcoma; Survival Rate; TP53 gene; Techniques; Toxic effect; Transcription Repressor/Corepressor; Treatment Protocols; United States; Work; aggressive therapy; base; chemotherapy; childhood sarcoma; clinically significant; cohort; disorder subtype; exome; falls; functional genomics; high risk; improved; in vitro Model; meetings; member; mortality; multimodality; neurofibroma; novel therapeutics; patient population; patient stratification; patient subsets; pre-clinical; prognostic; prognostic significance; prospective; response; sarcoma; therapeutic target; therapy development; tool; transcriptome sequencing; transcriptomics; tumor; user-friendly; whole genome

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 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, a more focused retrospective analysis is needed to determine the prognostic significance of the discovered mutations. In collaboration with the Children's Oncology Group, we have sequenced 675 clinically annotated rhabdomyosarcoma cases. In this project, we are using high-throughput capture based sequencing to determine the frequency with which the observed mutations occur and determine whether these mutations could be used as markers of prognosis or response to therapy. The specific objectives of the study are the following: 1. To validate our pilot study and accurately estimate the frequency of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in low, intermediate and high-risk embryonal rhabdomyosarcomas. 2. To estimate the frequency of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in a small cohort of alveolar histology tumors. 3. To determine the clinical significance of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in patients with central pathology confirmed ERMS cases. 4. To develop an accurate and reproducible assay for assessing mutations in NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 that can be used in prospective clinical trials. This year we have validated that our assay can reliably detect mutations even at low frequency and that we can accurately detect gene copy number changes. We have developed a bioinformatics pipeline and visualization portal that enables a user-friendly interface with the generated data. Clinical outcome correlation analysis has been performed in concert with the Childrens Oncology Group and we have demonstrated the clinical impact of the presence or absence of each genetic lesion. Mutations of MYOD1 and TP53 were found to be associated with clinical outcome and 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 Childrens Oncology Group trials. 2. Efforts in Malignant Peripheral Nerve Sheath Tumor (MPNST) The goal of this work is to provide further understanding of the genetic and transcriptomic mechanisms of the tumor evolution underlying the transformation of a Neurofibroma to a Malignant Peripheral Nerve Sheath Tumor (MPNST). 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 have built inducible in vitro model systems and performed ChIPseq, RNAseq and ATACseq to nominate genes whose expression is altered upon loss of these genes. These preclinical tools have allowed us to identify genes downstream of PRC2 loss that are potential therapeutic targets. We further interrogate these genes for functional relevance to MPNST using biochemical, molecular biology and CRISPR technologies.

1.横纹肌肉瘤（RMS）是一种肌源性癌症，是儿童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至70 - 80%，尽管具有显著的毒性。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%。治疗分配目前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的患者（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%），并且具有异质性临床结局。这表明，这些儿童中的一些可以用不那么积极的治疗方法治疗，或者应该被认为患有更具侵略性的疾病。为了进一步表征这种肿瘤类型的遗传事件，我们的小组与儿童肿瘤组合作，使用全基因组、全外显子组和全转录组测序的组合以及高分辨率SNP阵列沿着进行了大规模测序工作，以表征147个肿瘤/正常对中的体细胞改变的景观。我们的研究结果描述了一组异质性遗传事件似乎驱动RMS，最显著的是肺泡亚型中的PAX 3/7-FOXO 1融合和融合阴性肿瘤中多个RAS通路基因的突变，包括10个癌症共有基因（NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF 1、FGFR 4、FBXW7、CTNNB 1）中的复发性遗传病变。虽然这些突变中的大多数似乎是相互排斥的，但肿瘤的一个子集似乎在同一肿瘤内具有共存的病变;这可能表明这些肿瘤中的生物学相关进展。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，需要进行更集中的回顾性分析，以确定发现的突变的预后意义。在与儿童肿瘤组的合作中，我们对675例临床注释的横纹肌肉瘤病例进行了测序。在这个项目中，我们使用基于高通量捕获的测序来确定观察到的突变发生的频率，并确定这些突变是否可以用作预后或治疗反应的标志物。本研究的具体目标如下：1.验证我们的初步研究，并准确估计低、中、高危胚胎性横纹肌肉瘤中NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF 1、FGFR 4、FBXW7、CTNNB1突变的频率。2.在一个小的肺泡组织学肿瘤队列中估计NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF 1、FGFR 4、FBXW7、CTNNB1突变的频率。3.确定中心病理学证实的ERMS病例患者中NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF 1、FGFR 4、FBXW 7、CTNNB 1突变的临床意义。4.开发可用于前瞻性临床试验的NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF 1、FGFR 4、FBXW7、CTNNB1突变评估的准确且可重复的检测方法。今年，我们已经验证了我们的检测方法即使在低频率下也能可靠地检测突变，并且我们可以准确地检测基因拷贝数的变化。我们开发了一个生物信息学管道和可视化门户，可以为生成的数据提供用户友好的界面。临床结果相关性分析已经与儿童肿瘤组合作进行，我们已经证明了每种遗传病变的存在或不存在的临床影响。MYOD 1和TP 53的突变被发现与临床结果相关，这些结果在美国临床肿瘤学会年会和儿童肿瘤学小组年会上发表。目前的努力集中在使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，以集中描述所有参加儿童肿瘤组试验的横纹肌肉瘤患者。2.恶性外周神经鞘瘤（MPNST）的努力这项工作的目标是提供进一步了解肿瘤演变的遗传和转录机制的神经纤维瘤转化为恶性外周神经鞘瘤（MPNST）。利用儿科肿瘤学分支内的专业知识和患者群体，在该项目中，我们使用临床前模型系统来剖析NF1肿瘤从良性前体向侵袭性MPNST转变时发生的遗传和表观遗传变化。具体来说，我们正在研究突变或删除的影响，在一个polycomb复合体（PRC2）的成员SUZ12或EED的最后一步，在转化为MPNST。PRC2复合物是细胞内的主要转录抑制物，并且在来自患者样品的基因组测序研究中已经发现了该复合物的成员的反复改变。在本财政年度内，我们建立了诱导型体外模型系统，并进行了ChIPseq、RNAseq和ATACseq，以确定在这些基因丢失后表达发生改变的基因。这些临床前工具使我们能够识别PRC2缺失下游的基因，这些基因是潜在的治疗靶点。我们使用生物化学，分子生物学和CRISPR技术进一步询问这些基因与MPNST的功能相关性。