High-resolution analysis of structural genomic alterations in neuroblastoma by linked-read whole-genome sequencing

通过连接读全基因组测序对神经母细胞瘤的结构基因组改变进行高分辨率分析

• 批准号: 433152224
• 负责人: Dr. Christoph Bartenhagen
• 金额: --
• 依托单位: Abteilung Pädiatrische molekulare Onkologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/433152224?language=en
• 关键词: resolution; analysis; structural; genomic; alterations

Neuroblastoma is a malignant solid tumor of childhood with a broad range of clinical courses. In roughly half of the patients, outcome is excellent with no or limited cytotoxic treatment, due to spontaneous regression of the tumor. By contrast, fatal outcome occurs frequently in the remaining cases, despite intensive multimodal treatment. We recently demonstrated that high-risk neuroblastoma is defined by the presence of telomere maintenance, whereas such mechanisms are absent in spontaneously regressing cases (Peifer et al., Nature 2015; Ackermann et al., Science 2018). In addition, genomic alterations of RAS and p53 pathway genes contribute to malignant transformation in roughly one-third of high-risk cases. It has remained unclear, however, which genes drive tumorigenesis in the remaining two-thirds of these tumors. The paucity of single nucleotide variants in cancer-related genes identified by previous studies along with the frequent occurrence of segmental chromosomal copy number alterations suggest that structural genomic alterations may play a significant role in the pathogenesis of high-risk neuroblastoma. We hypothesize that the relevance of structural alterations for the pathogenesis of high-risk neuroblastoma has been underestimated in the past. Linked-read whole-genome sequencing (WGS) provides a novel and valuable tool to accurately determine haplotypes over megabase regions of the genome, which is a prerequisite for in-depth reconstruction of complex genomic rearrangements. In the proposed study, we are therefore aiming to discover pathogenetically relevant structural alterations in high-risk neuroblastoma by linked-read sequencing of primary tumors, which will be supplemented by information on gene expression profiles, histone marks, and clinical data of the corresponding patients. To this end, we will perform linked-read WGS of 50 primary high-risk neuroblastomas and matched normal controls, as well as relapse samples of 20 of these cases. We will develop and optimize computational analysis pipelines for linked-read WGS data to facilitate reconstruction of complex structural genomic alterations occurring in cancer cells. In addition, we will assess the effect of such alterations on gene expression profiles and histone modifications by RNA sequencing and chromatin immunoprecipitation coupled to sequencing, respectively. We will also integrate results from sequencing data with clinical information of the same patients to determine potential consequences of structural variants on the clinical phenotype of the disease. Finally, associations of structural alterations with clinical variables will be validated in an independent cohort using fluorescence in situ hybridization analysis. Together, we expect that our studies will provide profound novel insights into the genetic etiology of high-risk neuroblastoma, which forms the basis for establishing biomarker-guided personalized treatment strategies in this deadly malignancy.

神经母细胞瘤是一种发生于儿童的恶性实体瘤，临床病程广泛。在大约一半的患者中，由于肿瘤的自发消退，没有或有限的细胞毒性治疗的结果是极好的。相比之下，致死性结局在其余病例中频繁发生，尽管进行了强化的多模式治疗。我们最近证明，高危神经母细胞瘤是由端粒维持的存在定义的，而在自发消退的病例中不存在这种机制（Peifer et al.，Nature 2015; Ackermann等人，Science 2018）。此外，RAS和p53通路基因的基因组改变导致大约三分之一的高危病例发生恶性转化。然而，目前尚不清楚哪些基因驱动其余三分之二的肿瘤发生。沿着以往研究发现的肿瘤相关基因中单核苷酸变异的缺乏以及染色体片段拷贝数的频繁改变提示结构性基因组改变可能在高危神经母细胞瘤的发病机制中起重要作用。我们推测，在过去，结构改变与高危神经母细胞瘤发病机制的相关性被低估了。连接阅读全基因组测序（Linked-read whole-genome sequencing，WGS）提供了一种新的、有价值的工具来准确地确定基因组大区域的单倍型，这是深入重建复杂基因组重排的先决条件。因此，在拟议的研究中，我们的目标是通过原发性肿瘤的连接阅读测序来发现高危神经母细胞瘤中的病理相关结构改变，并通过相应患者的基因表达谱，组蛋白标记和临床数据信息进行补充。为此，我们将对50例原发性高危神经母细胞瘤和匹配的正常对照以及其中20例复发病例的样本进行连锁阅读WGS。我们将开发和优化连接读取WGS数据的计算分析管道，以促进癌细胞中发生的复杂结构基因组改变的重建。此外，我们将评估这种改变对基因表达谱和组蛋白修饰的影响，分别通过RNA测序和染色质免疫沉淀结合测序。我们还将整合测序数据的结果与相同患者的临床信息，以确定结构变异对疾病临床表型的潜在影响。最后，将使用荧光原位杂交分析在独立队列中验证结构改变与临床变量的关联。总之，我们期望我们的研究将为高危神经母细胞瘤的遗传病因学提供深刻的新见解，这为在这种致命的恶性肿瘤中建立生物标志物指导的个性化治疗策略奠定了基础。