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

Abstract

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, DKK1, 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. Key words: Medulloblastoma; Molecular subtype; Child; Adolescent; Polymerase chain reaction; Immunochemistry