Genomic characterization and development of therapies for Rhabdomyosarcoma

横纹肌肉瘤的基因组特征和治疗方法的开发

• 批准号: 9344062
• 负责人: John Shern
• 金额: $ 20.6万
• 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/9344062
• 关键词: Accounting; Alveolar; Asses; Bioinformatics; Biological Assay; CTNNB1 gene; Categories; Cell Line; Chemotherapy-Oncologic Procedure; Child; Childhood Rhabdomyosarcoma; Childhood Soft Tissue Sarcoma; Clinical; Clinical Trials; Collaborations; Consensus; Data; Data Analyses; Data Set; Development; Diagnosis; Disease; Dose; Embryonal Rhabdomyosarcoma; Event; FBXW7 gene; FGFR4 gene; FOXO1A gene; Frequencies; Gene Dosage; Gene Targeting; Genes; Genetic; Genomics; HRAS gene; High-Throughput Nucleotide Sequencing; Histology; KRAS2 gene; Knowledge; Lead; Lesion; Localized Disease; Malignant Neoplasms; Measures; Molecular Genetics; Mutation; NF1 gene; NRAS gene; Natural Products; Nucleotides; Oncogenes; Oncogenic; Outcome; Output; PAX3 gene; PIK3CA gene; Pathology; Pathway interactions; Patients; Pediatric Oncology Group; Pilot Projects; Preclinical Drug Evaluation; Prognostic Marker; Recurrence; Relapse; Resolution; Rhabdomyosarcoma; Risk; SNP array; Sequence Analysis; Subgroup; Survival Rate; TP53 gene; Toxic effect; aggressive therapy; base; clinically significant; cohort; exome; falls; fusion gene; high risk; improved; mortality; next generation sequencing; novel therapeutics; patient stratification; prognostic; prognostic significance; prospective; response; therapy development; transcription factor; transcriptome sequencing; tumor; whole genome

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 162 cases to date. 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.

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到70%-80%，尽管有显著的毒性。不幸的是，尽管进行了积极的治疗，转移性疾病患者的5年存活率仍然只有30%。目前的治疗方案是基于临床病理特征，使用这些标准，可以识别三个不同的亚组患者(低、中和高风险)。然而，许多患者属于中等风险类别(约占所有患者的50%)，临床结果各不相同。这表明，这些儿童中的一些可以接受不那么激进的治疗，或者应该被认为患有更具侵袭性的疾病。为了进一步表征这种肿瘤类型背后的遗传事件，我们的团队与儿童肿瘤学小组合作，进行了一项大规模的测序工作，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阵列来表征147对肿瘤/正常对的体细胞变化情况。我们的发现描述了一组异质性的遗传事件似乎最明显地驱动RMS的是肺泡型的PAX3/7-FOXO1融合和融合阴性肿瘤中多个RAS途径基因的突变，包括10个癌症共同基因(NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1)中的复发遗传性病变。虽然这些突变中的大多数似乎是相互排斥的，但部分肿瘤似乎在同一肿瘤内存在共存的病变；这可能表明这些肿瘤的生物学相关进展。不幸的是，这些病例的大部分临床注释是不完整的，严重限制了我们从这个数据集获得预后信息的能力。为了克服这一点，需要更有针对性的回顾分析来确定所发现的突变的预后意义。与儿童肿瘤学小组合作，我们确定了675例有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。本研究的具体目标如下：1.验证我们的初步研究，准确估计NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1突变在低、中、高危胚胎性横纹肌肉瘤中的频率。2.研究NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1基因在肺泡组织学肿瘤中的突变频率。3.探讨中枢病理证实的ERMS患者NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1基因突变的临床意义。4.建立一种准确、重复性好的检测NRAS、KRAS、HRAS、PIK3CA、BCOR、TP53、NF1、FGFR4、FBXW7、CTNNB1基因突变的方法，用于前瞻性临床试验。在过去的一年里，我们建立了一条提取核苷酸、有针对性的基因捕获和测序以及数据的生物信息学分析的管道。我们已经验证了我们的检测方法即使在低频率下也可以可靠地检测到突变，并且我们可以准确地检测到基因拷贝数的变化。到目前为止，我们已经完成了162例病例的测序和数据分析。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PAX3-FOXO1的新疗法。Pax3-FOXO1是一种与特别糟糕的临床结果相关的基因变化。在过去的一年里，我们已经建立和优化了一种细胞系结构，可以稳健地测量PAX3-FOXO1融合基因的转录输出。我们已经完成了大约125,000种化合物和天然产品提取物的初步筛选。我们的细胞系构建在实验中被发现表现可靠，生成的数据已经得到验证。从初步筛选中，我们鉴定了大约200个候选化合物或混合物，它们以剂量反应的方式特异性地抑制PAX3-FOXO1的转录输出。我们已经开发了一种二级筛查，可以快速评估融合基因的其他下游目标，并使用RNA测序来全面表征我们的主要候选化合物的转录效应。