A pilot study of DNA methylation in pediatric germ cell tumors

描述（由申请人提供）： 儿科生殖细胞肿瘤（GCT）是一组异质性肿瘤，被假设为发生在子宫内的事件的结果，虽然病因仍在很大程度上未知。异常DNA甲基化与多种类型癌症的病因学有关，由于在正常发育期间在生殖系和早期胚胎中发生的广泛的表观遗传重编程，其在GCT中具有特别相关的潜力。有限的证据支持表观遗传学改变在GCT发展中的潜在作用;然而，迄今为止尚未对儿科GCT中甲基化模式的改变进行全面研究。我们的长期目标是了解表观遗传学改变在儿科GCT中的作用，以及这些改变如何受到环境暴露的影响，目的是在儿童肿瘤组中建立一个大型的儿科GCT遗传流行病学研究。本试验的主要目的是从58例儿童GCT（包括生殖细胞瘤、卵黄囊瘤、畸胎瘤和混合性生殖细胞瘤）中提取DNA，并按组织学类型确定一组启动子高甲基化改变的基因。我们的假设是，以前参与致癌作用的基因中的DNA甲基化在肿瘤和正常组织之间以及在肿瘤组织学上是不同的。为了探索这一假设，将评价以下特定目的：1）鉴定儿科GCT中具有改变的启动子高甲基化的基因，2）通过肿瘤组织学探索DNA甲基化的肿瘤间和肿瘤内异质性。我们将使用两阶段方法评估DNA甲基化。首先，我们将使用Illumina GoldenGate甲基化癌症面板测量先前与癌症发生有关的807个基因的面板中的DNA甲基化，这将使我们能够选择具有GCT特征的CpG位点。然后，我们将验证和扩大前15个CpG位点的焦磷酸测序相同的样本。在完成拟议的研究时，我们期望我们将确定一组DNA甲基化改变的基因，这些基因可能与儿科GCT的发展相关，并且这些数据将作为初步数据纳入NIH R 01申请中，以收集额外的肿瘤和流行病学数据，用于儿科GCT的表观遗传学研究。本申请中提出的研究具有重要意义，因为它将更全面地了解整个基因组中癌症相关基因以及不同组织学亚型肿瘤中儿科GCT的DNA甲基化。 公共卫生相关性： 胎儿发育和生命早期的环境暴露被认为在儿童和成人癌症的发展中发挥作用，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），并且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提供一些见解如何早期生活暴露导致基因功能的长期改变，并可能提供重要的见解胎儿致癌的起源。这项试点资助的总体目标是评估儿科生殖细胞肿瘤的表观遗传学改变，这将作为这一重要且研究不足的癌症未来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.