Genomic and expression profiling of neuroblastoma

尽管近年来肿瘤治疗取得了很大进展，但晚期神经母细胞瘤患者的总体生存率仍然很低。此外，目前使用的预后标志物有时不足以预测中危型肿瘤患者的预后。本研究的主要目的是通过对神经母细胞瘤样本的基因组畸变和表达谱的分析，构建一个系统，以早期分类这种肿瘤，并帮助选择适当的治疗策略。1）神经母细胞瘤的基因表达谱我们制作了一个肿瘤特异性的cDNA芯片，其中包含了从原发性神经母细胞瘤中收集的11，000个基因。将该芯片应用于不同分化程度的神经母细胞瘤的表达谱分析。与此同时，我们与UCSF癌症中心合作，应用基于BAC阵列的阵列CGH方法来调查肿瘤DNA中发生的拷贝数变化。至c 关于我们 在11，000个cDNA芯片上，通过基因组丢失和/或获得来比较基因表达水平，通过在UCSC基因组浏览器数据库中搜索它们的核苷酸序列来进行11，000个cDNA的染色体作图。2）神经母细胞瘤基因组和分子标记的综合分析我们完成了268例原发性神经母细胞瘤和细胞系的阵列CGH分析，比较了基因表达标记和染色体丢失和获得等基因组畸变。我们可以定义染色体1 lq上缺失重叠的最小区域约为10 Mb，其中一个基因在有利和不利的神经母细胞瘤之间表现出差异表达。因此，我们进一步使用多个神经母细胞瘤RNA样本进行基因的定量实时RT-PCR分析，并通过亚硫酸氢盐测序检查其启动子区域的表观遗传修饰。对染色体1 p36区域进行类似的分析。我们的下一步计划是确认目前的结果，并对我们已经确定的候选基因进行功能分析。少

In spite of recent progress in cancer therapy, the overall survival rate of the patients with neuroblastoma in advanced stages is still quite low. Furthermore, prognostic markers currently used are sometimes not enough to predict prognosis of the patients with intermediate risk type of tumors. The main purpose of this study is to construct a system for early classification of such tumors and to help choose appropriate therapeutic strategy, through the analyses of genomic aberrations and expression profiling of neuroblastoma samples. In this study, following results were obtained :1) Expression profiling of neuroblastomaWe previously made an in-house, tumor-proper cDNA chip harboring the 11,000 genes collected from primary neuroblastomas. The chip was applied to the expression profiling of neuroblastomas with various prognoses. In parallel, by collaborating with UCSF Cancer Center, we applied BAC array-based array CGH method to survey the copy number changes occurred in tumor DNAs. To c … More ompare the gene expression level with genomic loss and/or gain of each gene on the 11,000 cDNA chip, chromosome mapping of 11000 cDNAs was conducted by searching their nucleotide sequences against the UCSC genome browser databases. The data obtained were submitted to the public microarray databases NCBI GEO.2) Integrated analyses of genomic and molecular signatures of neuroblastomaWe have finished array CGH analysis of 268 samples prepared from primary neuroblastomas and cell lines and compared the gene expression signatures and genomic aberrations such as chromosomal losses and gains. We could define the smallest region of overlaps of deletions at chromosome 1lq to be approximately 10 Mb, in which one gene exhibiting the differential expression between favorable and unfavorable neuroblastomas was identified. Therefore, we further performed quantitative real-time RT-PCR analysis of the gene by using multiple neuroblastoma RNA samples and also examined epigenetic modifications of its promoter region by bisulfite sequencing. Similar analysis was conducted for chromosome 1p36 region. Our next plan is to confirm the present results and do the functional analyses of the candidate genes that we have identified. Less