Microarray analysis for detecting biological characteristics in neuroblastoma

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DNA samples of neuroblastoma tissues were examined CGH array using bacterial artificial chromosomes derived from human chromosome 1, 2, 3, 11, 13, 17, 19, 21, 22. Alterations of signals, especially chromosomes 1, 11 and 17, were detected in unfavorable neuroblastomas but not in favorable tumors. Analysis of cDNA microarray using approximately 6500 clones derived from embryonal brain revealed 43 upregulated genes in unfavorable tumors, 20 and 32 upregulated genes in regressing and maturing tumors, respectively. On the other hand, microsort analysis revealed 63 and 39 genes upregulated in unfavorable and favorable tumors, respectively. Real time quantitative RT-PCR in the upregulated genes identified by above technologies identified 21 known genes including MYCN, hTERT, VGEF, Cyclin G1, CD44, NGF, and Caspase 8. In the "composite type" tumor tissues which have heterogeneity of tumor findings in the same tumors, we collected tumor cells in the part with highly dismorphologic findings or in the part with telomerase positive cells by microdissection technique. And we also microdissected the viable tumor cells remained in the postchemotherapeutic tumor sections. Then, total RNA isolated from these collected cells was amplified and then used for microarray. These results identified 23 genes upreglated in the highly malignant cells. Almost all genes were included in the genes identified in the unfavorable tumors. Regressing neuroblastomas showed the up-regulation of apoptosis-relating genes such as caspase 8 and 9 and maturing tumors showed the up-regulation of the neuronal differentiating factors such as NTRK1 and NGF. The comparison between telomerase upregulating tumor and others revealed 102 different expressing genes including hsp 90 and Rad 50, which may directly regulate telomerase. These results suggested that the useful markers for classification of neuroblastoma are the expression of 21 genes and gene dosage of chromosome 1, 11, and 17.