实时荧光定量PCR确定儿童和青少年髓母细胞瘤分子亚型的准确性分析  被引量:3

Accuracy analysis of identifying molecular subtypes of children and adolescents with medulloblastoma using real-time fluorescence quantitative PCR

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作  者:张晶[1] 李春德[2] 刘丕楠[2] 李朋[2] 李仕维[2] 张顺[2] 赵赤[2] 赵赋[1] 

机构地区:[1]首都医科大学,北京市神经外科研究所,100050 [2]首都医科大学附属北京天坛医院神经外科

出  处:《中华神经外科杂志》2016年第9期867-872,共6页Chinese Journal of Neurosurgery

基  金:北京市卫生系统高层次卫生技术人才培养计划(2014-2-008);首都卫生发展科研专项(2011-1015-01)

摘  要:目的探讨实时荧光定量PCR方法(RT-qPCR)诊断儿童和青少年髓母细胞瘤分子亚型的可靠性。方法回顾性纳入首都医科大学附属北京天坛医院神经外科2011年1月至2015年12月经病理学确诊为髓母细胞瘤的患者30例。采用RT—qPCR方法对肿瘤的冰冻样本进行检测,选取11个分子亚型特征性标记基因(OTX2、FOXG1B、MYC、DKK1、WFJ、DKK2、MYCN、SFRPl、HHIP、OTX1、NPR3),依据基因表达水平差异进行无监督聚类分析和微列阵预测分析,并绘制热图和重心图;结合表达谱芯片和免疫组化方法进行验证,采用单因素方差分析分子亚型间标记基因表达值的差异,分析分子亚型临床因素分布的差异。结果采用RT—qPCR方法可将30例髓母细胞瘤患者分成4个分子亚型,其中SHH亚型17例,WNT亚型4例,Group3亚型4例,Group4亚型5例,分型结果与免疫组织化学染色结果和无监督聚类分析结果完全一致;在各个亚型间,11个特征性标记基因的表达差异均有统计学意义(P〈0.01或P〈0.05),其中SFRP1、MYCN、NPR3及MYC是区别亚型的关键基因;SHH亚型和WNT亚型在性别分布上(男:女分别为4.7:1.0和1:3)、诊断年龄上(≤3岁:3~16岁分别为1.0:4.7和3:1),差异均有统计学意义(均P=0.022)。结论RT-qPCR方法能准确确定儿童和青少年髓母细胞瘤的4个分子亚型。Objective To investigate the reliability of diagnosing the molecular subtypes of children and adolescents with medulloblastoma (MB) using real-time fluorescence quantitative PCR (RT-qPCR). Methods Thirty patients with MB diagnosed by pathology and admitted to the Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University from January 2011 to December 2015 were enrolled retrospectively. RT-qPCR was used to detect the frozen samples of MB. Eleven subgroup-specific protein-coding genes (OTX2, FOXG1B, MYC, DKKI, WIF1, DKK2, MYCN, SFRP1, HHIP, OTX1, and NPR3 ) were selected. Unsupervised hierarchical cluster analysis and prediction analysis of microarray were conducted according to the differences of gene expression levels, and the heat map and gravity map were drawn. The result of subgroup was validated by immunohistochemical method and expression profile. One-way ANOVA was used analyze the differences of the marker gene expression value among the molecular subtypes. The differences of the clinical factor distribution of the molecular subtypes were analyzed. Results Thirty patients with MB were divided into 4 molecular subtypes by using RT-qPCR, including SHH subtype (n = 17), WNT subtype (n =4), Group 3 subytype (n =4), and Group 4 subtype ( n = 5). The results of grouping, immunohistochemical staining and unsupervised clustering analysis were completely consistent. There were significant differences in 11 subgroup-specific protein-coding genes expression among the subgroups (P〈0.01 or P〈0.05). Among them, SFRP1, MYCN, NPR3, and MYC were the key genes for distinguishing subtypes. There were significant differences in the sex distribution (male:female, 4. 7 : 1.0 and 1:3 respectively) and age at diagnosis (≤3 year: 3 - 16, 1.0:4.7 and 3 : 1 respectively) between the SHH subtype and the WNT subtype ( all P = 0. 022). Conclusion RT-qPCR may accurately identify the 4 molecular subtypes in children and adolescents with MB.

关 键 词:髓母细胞瘤 分子亚型 儿童 青少年 聚合酶链反应 免疫组织化学 

分 类 号:R739.4[医药卫生—肿瘤]

 

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