Genomic characterization and development of therapies for pediatric sarcoma

1. 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小组合作，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，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。我们与儿童肿瘤组合作，对675例临床注释横纹肌肉瘤病例进行了测序。在这个项目中，我们正在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记。本研究的具体目标如下：1。验证我们的前期研究，准确估计低、中、高风险胚胎性横纹肌肉瘤中NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1突变的频率。2. 估计NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1突变在肺泡组织学肿瘤小队列中的频率。3. 目的探讨NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1突变在中枢性病理确诊的ERMS患者中的临床意义。4. 开发一种准确、可重复的检测方法，用于评估NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1的突变，可用于前瞻性临床试验。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已与儿童肿瘤组合作进行，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，我们发现MYOD1和TP53突变与融合阴性横纹肌肉瘤的临床预后相关，这些结果已在美国临床肿瘤学会年会上以及儿童肿瘤学会年会上公布，并提交发表。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。此外，我们对中高风险临床试验收集的回顾性样本进行了额外的测序，为目前正在开发的临床试验提供基础数据，这些临床试验将使用遗传标记来改进临床风险分层和临床决策。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）转化的肿瘤进化的遗传和转录机制。mpnst是一种毁灭性的风险，主要影响NF1基因（1型神经纤维瘤病）的种系突变患者。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。

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. 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 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. In the current year, we have defined that mutations of MYOD1 and TP53 were found to be associated with clinical outcome in fusion negative Rhabdomyosarcoma and these results were presented at the American Society of Clinical Oncology Annual Meeting as well as the Childrens Oncology Group annual meeting and this data is submitted for publication. 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. In additional we performed additional sequencing of retrospective samples collected on intermediate and high risk clinical trials to provide foundational data for clinical trials currently under development that will use genetic markers for improved clinical risk stratification and clinical decision making. 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 Plexiform Neurofibroma to a Malignant Peripheral Nerve Sheath Tumor (MPNST). MPNSTs are a devastating risk that predominantly affects patients with a germline mutation in the gene NF1 (Neurofibromatosis Type 1). 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.