Microarray analysis for detecting biological characteristics in neuroblastoma

对322例人类神经母细胞瘤病例进行了微阵列和基于阵列的CGH分析。基于阵列的CGH阵列显示MYCN扩增，染色体1、3、4、11、14和17出现畸变。用于检测这些畸变的定制CGH阵列显示，染色体11q缺失是一个独立的不良预后相关因素。我们还通过cDNA微阵列、全基因组寡基因微阵列和微排序技术选择182个预后相关基因进行定制阵列。使用这种自定义阵列进行表达分析，预测神经母细胞瘤的预后具有很高的准确性（95%）。我们还制作了用于检测hTERT、caspases 8和caspases 9启动子区域DNA甲基化的定制芯片。结果显示，只有2个caspase 8甲基化位点与预后不良相关。9例肿瘤内异质性，30例化疗后切除，11例多灶性肿瘤，8例复发，6例肿瘤退化或成熟，采用显微解剖分离更多细胞，比较各原发肿瘤的基因表达谱。12例表达谱发生极大改变的肿瘤均表现出较差的预后，而在多灶性肿瘤或退化/成熟肿瘤中，即使DNA倍体模式不同，也没有肿瘤表现出显著的基因表达改变。在12株神经母细胞瘤细胞株中，端粒酶抑制剂或小干扰RNA沉默hTERT分别导致5株和7株细胞凋亡和生长抑制。在这些细胞系中，基因表达分析显示细胞生长信号减少和凋亡相关基因激活。维甲酸诱导分化的3株细胞MYCN表达和端粒酶表达降低，凋亡相关信号和神经元分化增加。这种带有CGH分析的定制阵列有助于评估每个神经母细胞瘤的特征。此外，它也可能是选择化疗方案和分化治疗的有用工具。少

Microarray and array-based CGH analyses were examined in 322 human neuroblastoma cases. Array-based CGH array revealed MYCN amplification, aberrations in chromosomes 1,3,4,11,14, and 17. Custom CGH array for detection of these aberrations revealed that chromosome 11q deletion was an independently poor prognosis-associated factor. We also made custom array of 182 prognosis-related genes selected by cDNA microarray, genome-wide oligomicroarray for gene expression, and microsort technology. Expression analysis using this custom array could exhibit high accuracy (95%) to predict the neuroblastoma prognosis. We also made custom microarray for detecting DNA methylation in the promoter regions of hTERT, caspases 8 and 9. It revealed that only 2 methylated loci of caspase 8 were correlated with poor prognosis. In 9 cases with intratumoral heterogeneity, 30 tumors resected after chemotherapy, 11 multifocal tumors, and 8 recurrent tumors, 6 tumors which consequently regressed or matured, tumor c … More ells were isolated using microdissection to compare the gene expression profiling with each primary tumor. All 12 tumors whose expression profilings were extremely altered showed poor outcome, while no tumor in multifocal tumors or regressing/maturing tumors showed remarkable alteration of gene expression even if DNA ploidy patterns were different.Telomerase inhibitor or silencing of hTERT by small interfering RNA in 12 neuroblastoma cell lines resulted that 5 or 7 cell lines showed apoptosis and growth inhibition, respectively. In these cell lines, gene expression analysis revealed reduction of cell growth signals and activation of apoptosis related genes. Moreover, 3 cell lines which are induced differentiation by retinoic acid showed decrease of MYCN expression and telomerase expression and increase of apoptosis related signals and neuronal differentiation.This custom array with CGH analysis is useful to evaluate characteristics in each neuroblastoma. Moreover, it may be also a useful tool to choice the chemotherapeutic regimen and differentiation therapy. Less