Alaminos Miguel, Davalos Veronica, Cheung Nai-Kong V, Gerald William L, Esteller Manel
Cancer Epigenetics Laboratory, Spanish National Cancer Centre (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
J Natl Cancer Inst. 2004 Aug 18;96(16):1208-19. doi: 10.1093/jnci/djh224.
Neuroblastoma is the most common extracranial solid malignancy in infancy and childhood, but the biological factors involved in its development and progression are still unclear. Transcriptional silencing of tumor suppressor genes mediated by hypermethylation of promoter CpG islands is a hallmark of human tumors. We addressed the clinical relevance of promoter hypermethylation in neuroblastoma.
We examined the methylation status of 45 candidate genes representative of many cellular pathways in 10 neuroblastoma cell lines and of 10 of these genes in 145 tumor samples (118 of them were primary neuroblastomas). We used Fisher's exact test to examine the association of CpG island methylation and clinical subgroups and Kaplan-Meier analysis to determine the association between methylation and survival in primary tumors. Cluster analysis was used to group cell lines and tumors by gene methylation status. Bonferroni-corrected statistical tests were two-sided.
Clustering of neuroblastoma cell lines on the basis of hypermethylation distinguished lines with MYCN amplification (a negative prognostic factor) from those without it (P =.012). Promoter hypermethylation of the developmental gene HOXA9 was associated with mortality in noninfant patients (P =.04) and in tumors lacking MYCN amplification (P =.023). Hypermethylation of the proapoptotic gene TMS1 and the cell cycle gene CCND2 was associated with stage 4-progressing tumors (P<.001), but the genes were never methylated in stage 4S tumors, which undergo spontaneous regression. Hypermethylation of the differentiation gene RARbeta2 was associated with patient survival (P =.032). Unsupervised hierarchical cluster analysis of all tumors based on methylation of the 10 genes separated several clinically relevant groups of tumors.
Profiling the status of CpG island hypermethylation in human primary neuroblastomas may have clinicopathologic value.
神经母细胞瘤是婴幼儿和儿童期最常见的颅外实体恶性肿瘤，但其发生发展过程中涉及的生物学因素仍不清楚。启动子CpG岛高甲基化介导的肿瘤抑制基因转录沉默是人类肿瘤的一个标志。我们探讨了神经母细胞瘤中启动子高甲基化的临床相关性。
我们检测了10种神经母细胞瘤细胞系中代表多种细胞通路的45个候选基因的甲基化状态，以及145个肿瘤样本（其中118个为原发性神经母细胞瘤）中10个基因的甲基化状态。我们使用Fisher精确检验来检测CpG岛甲基化与临床亚组的相关性，并使用Kaplan-Meier分析来确定原发性肿瘤中甲基化与生存的相关性。聚类分析用于根据基因甲基化状态对细胞系和肿瘤进行分组。Bonferroni校正的统计检验为双侧检验。
基于高甲基化对神经母细胞瘤细胞系进行聚类，可区分出有MYCN扩增（一种不良预后因素）的细胞系和无MYCN扩增的细胞系（P = 0.012）。发育基因HOXA9的启动子高甲基化与非婴儿患者的死亡率相关（P = 0.04），与缺乏MYCN扩增的肿瘤的死亡率相关（P = 0.023）。促凋亡基因TMS1和细胞周期基因CCND2的高甲基化与4期进展性肿瘤相关（P<0.001），但在4S期肿瘤（可自发消退）中这些基因从未发生甲基化。分化基因RARbeta2的高甲基化与患者生存相关（P = 0.032）。基于这10个基因的甲基化对所有肿瘤进行无监督层次聚类分析，分离出了几个具有临床相关性的肿瘤组。
分析人类原发性神经母细胞瘤中CpG岛高甲基化状态可能具有临床病理价值。
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