Genomic characterization and development of therapies for Rhabdomyosarcoma

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、PIK 3CA、BCOR、TP 53、NF 1、FGFR 4、FBXW 7、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。我们与儿童肿瘤学小组合作，确定了675例临床注释的横纹肌肉瘤病例。在这个项目中，我们使用高通量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、PIK 3CA、BCOR、TP 53、NF 1、FGFR 4、FBXW 7、CTNNB 1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、PIK 3CA、BCOR、TP 53、NF 1、FGFR 4、FBXW 7、CTNNB 1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、PIK 3CA、BCOR、TP 53、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、PIK 3CA、BCOR、TP 53、NF 1、FGFR 4、FBXW 7、CTNNB 1突变评估的准确且可重复的检测方法。在过去的一年里，我们已经建立了一个用于提取核苷酸、靶向基因捕获和测序以及数据生物信息学分析的管道。我们已经验证了我们的检测方法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。迄今为止，我们已经完成了300例病例的测序和数据分析。我们已经开发了一个生物信息学管道和可视化门户，使用户友好的界面与生成的数据。我们已经启动了临床结果与存在或不存在每个遗传病变的相关性。在该项目的第二个目标中，我们正在使用高通量药物筛选来发现特异性抑制融合癌基因PAX 3-FOXO 1的新疗法。PAX 3-FOXO 1是一种与特别糟糕的临床结果相关的遗传变化。在过去的一年中，我们已经建立和优化了一个细胞系构建，可以稳健地测量PAX 3-FOXO 1融合基因的转录输出。我们已经完成了约125，000种化合物和天然产物提取物的初步筛选。发现我们的细胞系构建体在测定中可靠地运行，并且生成的数据已得到验证。从初步筛选中，我们鉴定了大约200种以剂量响应方式特异性抑制PAX 3-FOXO 1转录输出的候选化合物或混合物。我们已经开发了一种二次筛选，可以快速评估融合基因的其他下游靶点，并使用RNA测序来全面表征我们的先导候选化合物的转录效应。我们目前的功能特点是三个铅化合物的屏幕使用在体外和体内模型横纹肌肉瘤。

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 identified 675 clinically annotated rhabdomyosarcoma cases. In this project we are using high throughput 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. Within the past year, we have built a pipeline for the extraction of nucleotides, targeted gene capture and sequencing and bioinformatic analysis of the data. 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 completed sequencing and data analysis of 300 cases to date. We have developed a bioinformatics pipeline and visualization portal that enables a user friendly interface with the generated data. We have initiated clinical outcome correlations with the presence or absence of each genetic lesion. In the second objective of the project, we are using high-throughput drug screening to discover novel therapeutics that specifically inhibit the fusion oncogene PAX3-FOXO1. PAX3-FOXO1 is a genetic change that is associated with particularly bad clinical outcomes. Within the past year, we have built and optimized a cell line construct that can robustly measure the transcriptional output of the PAX3-FOXO1 fusion gene. We have completed the primary screen of approximately 125,000 compounds and natural product extracts. Our cell line construct was found to perform reliably in the assay and the generated data has been verified. From the primary screen we identified approximately 200 candidate compounds or mixtures that specifically inhibit the transcriptional output of PAX3-FOXO1 in a dose response manner. We have developed a secondary screen that can rapidly asses additional downstream targets of the fusion gene and have used RNA sequencing to globally characterize the transcriptional effects of our lead candidate compounds. We are currently functionally characterized three lead compounds from the screen using in vitro and in vivo models of rhabdomyosarcoma.