A pilot study of DNA methylation in pediatric germ cell tumors

描述(由申请人提供)： 儿童生殖细胞肿瘤(GCTS)是一组异质性肿瘤，被认为是由于宫内事件而发生的，尽管其病因大部分仍不清楚。异常的DNA甲基化与多种癌症的病因有关，由于在胚系和正常发育的早期胚胎中发生了广泛的表观遗传重编程，因此在GCTS中具有特别相关的潜力。有有限的证据支持表观遗传改变在GCTS发展中的潜在作用；然而，迄今为止还没有对儿科GCTS的甲基化模式改变进行全面的研究。我们的长期目标是了解表观遗传改变在儿童GCT中的作用以及这些改变可能如何受到环境暴露的影响，目的是在儿童肿瘤学小组中建立一项关于儿童GCT的大型遗传流行病学研究。这项试验的主要目标是识别一组从58例儿童GCT中提取的DNA启动子高甲基化改变的基因，包括生殖细胞瘤、卵黄囊肿瘤、畸胎瘤和混合生殖细胞肿瘤，总体上和按组织类型分类。我们的假设是，以前与癌症发生有关的基因中的DNA甲基化在肿瘤和正常组织之间以及肿瘤组织学上都是不同的。为了探索这一假说，将评估以下特定目标：1)在儿童GCTS中识别启动子高甲基化改变的基因；2)通过肿瘤组织学探索DNA甲基化在肿瘤内和肿瘤内的异质性。我们将使用两个阶段的方法评估DNA甲基化。首先，我们将使用Illumina GoldenGate甲基化癌症小组来测量之前与癌症发生有关的807个基因的DNA甲基化，这将使我们能够选择GCTS特有的CpG位点。然后，我们将通过对相同样本进行焦磷酸测序来验证和扩展前15个CpG位点。在拟议的研究完成后，我们期望我们已经确定了一组DNA甲基化改变的基因，这些基因可能与儿童GCT的发展相关，这些数据将作为初步数据包括在NIH R01应用程序中，以收集更多的肿瘤和流行病学数据，用于儿童GCT的表观遗传学研究。在这项申请中提出的研究具有重要意义，因为它将提供更全面的了解，在儿童GCT中，在整个基因组中的癌症相关基因以及不同组织亚型的肿瘤中的DNA甲基化。 公共卫生相关性：胎儿发育和早期生活中的环境暴露被认为在儿童和成人的癌症发展中发挥作用，尽管这些暴露影响疾病风险的机制尚不清楚。广泛的表观遗传重编程在受精后立即发生(在生殖细胞中)，这些表观遗传修饰在有丝分裂细胞分裂的整个生命周期中通常是稳定的。更好地了解这些表观遗传变化可能会为早期生命暴露如何导致基因功能的长期变化提供一些见解，并可能为癌症发生的胎儿起源提供重要的见解。这项试点拨款的总体目标是评估儿科生殖细胞肿瘤的表观遗传学变化，这将被用作未来对这种重要的、调查不足的癌症进行流行病学研究的试点数据。

DESCRIPTION (provided by applicant): Pediatric germ cell tumors (GCTs) are a heterogeneous group of tumors that are hypothesized to occur as a result of events in utero, although the etiology remains largely unknown. Aberrant DNA methylation, which has been implicated in the etiology of multiple types of cancer, has the potential to be especially relevant in GCTs due to the extensive epigenetic reprogramming that occurs in the germ line and early embryo during normal development. Limited evidence exists to support a potential role of epigenetic alterations in the development of GCTs; however, a comprehensive study of alterations in methylation patterns in pediatric GCTs has not been conducted to date. Our long term objective is to understand the role of epigenetic alterations in pediatric GCTs and how these alterations may be influenced by environmental exposures, with the goal of establishing a large genetic epidemiology study of pediatric GCT in the Children's Oncology Group. The primary objective for this pilot is to identify a panel of genes with altered promoter hypermethylation in DNA extracted from 58 pediatric GCTs, including germinomas, yolk sac tumors, teratomas and mixed germ cell tumors, overall and by histologic type. Our hypothesis is that DNA methylation in genes previously implicated in carcinogenesis will differ between tumor and normal tissue and also by tumor histology. To explore this hypothesis, the following specific aims will be evaluated: 1) identify genes with altered promoter hypermethylation in pediatric GCTs and 2) explore inter- and intratumoral heterogeneity in DNA methylation by tumor histology. We will evaluate DNA methylation using a two-staged approach. First, we will measure DNA methylation in a panel of 807 genes previously implicated in carcinogenesis using the Illumina GoldenGate Methylation Cancer Panel, which will allow us to select CpG sites that are characteristic of GCTs. We will then validate and expand the top15 CpG sites by Pyrosequencing on the same samples. At the completion of the proposed studies, it is our expectation that we will have identified a panel of genes with altered DNA methylation that are likely to be relevant in the development of pediatric GCT, and that these data will be included as preliminary data in an NIH R01 application to collect additional tumor and epidemiologic data for a study of the epigenetics of pediatric GCT. The research proposed in this application is significant because it will provide a more comprehensive understanding of DNA methylation in pediatric GCT in cancer-related genes throughout the genome as well as in tumors of different histologic subtypes. PUBLIC HEALTH RELEVANCE: Environmental exposures during fetal development and early life are thought to play a role in the development of cancer in both children and adults, although the mechanism by which these exposures influence disease risk is not clear. Extensive epigenetic reprogramming occurs immediately after fertilization (in the germ cells), and these epigenetic modifications are generally stable throughout the lifetime of mitotic cell divisions. A better understanding of these epigenetic changes may provide some insights on how early-life exposures lead to long-term alterations in gene function, and may provide important insights into the fetal origins of carcinogenesis. The overall objective of this pilot grant is to evaluate epigenetic alterations in pediatric germ cell tumors, which will be used as pilot data for a future epidemiologic study of this important and under- investigated cancer.