Expression Analysis of Juvenile Pilocytic Astrocytomas by Oligonucleotide Microarray Reveals Two Potential Subgroups. Homo sapiens

Juvenile pilocytic astrocytoma (JPA) is one of the most common brain tumors in children. The expression profiles of 21 JPAs, determined using Affymetrix GeneChip U133A, were compared with subjects with normal cerebella. The genes involved in neurogenesis, cell adhesion, synaptic transmission, central nervous system development, potassium ion transport, protein dephosphorylation, and cell differentiation were found to be significantly deregulated in JPA. These 21 JPAs were further clustered into two major groups by unsupervised hierarchical clustering using a set of 848 genes with high covariance (0.5-10). Supervised analysis with Significance Analysis of Microarrays software between these two potential subgroups identified a list of significant differentially expressed genes involved in cell adhesion, regulation of cell growth, cell motility, nerve ensheathment, and angiogenesis. Immunostaining of myelin basic protein on paraffin sections derived from 18 incompletely resected JPAs suggests that JPA without myelin basic protein–positively stained tumor cells may have a higher tendency to progress. Overall design: Under an Institutional Review Board–approved protocol, brain tumor tissues were obtained, after informed consent, from patients undergoing tumor resection or other tumor-related neurosurgical procedures at the Texas Children's Hospital, Baylor College of Medicine. Samples of normal cerebellar tissue (n = 2) were isolated from surgically removed tissue adjacent to resected tumor tissue. Portions of the tumors were fixed in 10% formaldehyde and embedded in paraffin for sectioning and pathologic reviews, and the residual tissue samples were snap-frozen in liquid nitrogen and stored at –80°C for RNA extraction. All samples were obtained at initial diagnosis with no prior exposure to chemotherapy or radiation therapy. Normal fetal brain RNA was obtained from Stratagene (La Jolla, CA) and a normal cerebellum RNA was from Ambion, Inc. (Austin, TX). Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) followed by DNase I treatment and clean-up on aRNeasy spin column (Qiagen, Valencia, CA). RNA quality and purity were assessed by agarose gel electrophoresis and absorbance measurement at A260/A280.

青少年毛细胞星形细胞瘤（Juvenile Pilocytic Astrocytoma, JPA）是儿童群体中最常见的脑肿瘤之一。本研究采用Affymetrix GeneChip U133A基因芯片，检测了21例JPA组织的基因表达谱，并以正常小脑组织作为对照样本进行比对分析。结果发现，JPA组织中涉及神经发生、细胞黏附、突触传递、中枢神经系统发育、钾离子转运、蛋白质去磷酸化以及细胞分化的基因均存在显著表达失调。本研究选取协方差介于0.5至10之间的848个基因，通过无监督层次聚类算法将这21例JPA样本划分为两大主要亚组。采用微阵列显著性分析（Significance Analysis of Microarrays）软件对这两个潜在亚组开展监督分析，筛选得到一系列显著差异表达基因，这些基因涉及细胞黏附、细胞生长调控、细胞运动、神经鞘形成及血管生成等生物学过程。对18例未完全切除的JPA患者的石蜡切片进行髓鞘碱性蛋白（Myelin Basic Protein, MBP）免疫染色结果显示，未检测到髓鞘碱性蛋白阳性肿瘤细胞的JPA病例，其肿瘤进展风险显著更高。

实验整体设计：本研究经机构审查委员会（Institutional Review Board, IRB）批准，所有受试者均签署书面知情同意书，样本采集自贝勒医学院德克萨斯儿童医院内，接受肿瘤切除术或其他肿瘤相关神经外科手术的患者的脑肿瘤组织。正常小脑组织样本（n=2）取自手术切除的肿瘤旁正常脑组织。将部分肿瘤组织以10%甲醛固定后进行石蜡包埋，用于切片制备与病理评估；剩余组织样本经液氮快速冷冻后，保存于-80℃冰箱，用于后续RNA提取。所有样本均采集自初诊患者，且所有患者在确诊前均未接受过任何化疗或放疗。正常胎儿脑组织RNA购自Stratagene公司（美国加利福尼亚州拉霍亚市），正常小脑组织RNA购自Ambion公司（美国得克萨斯州奥斯汀市）。总RNA提取采用Trizol试剂（Invitrogen公司，美国加利福尼亚州卡尔斯巴德市），随后进行DNase I消化，并通过RNeasy离心柱（Qiagen公司，美国加利福尼亚州瓦伦西亚市）进行纯化。RNA的质量与纯度通过琼脂糖凝胶电泳以及A260/A280吸光度比值进行检测与评估。