Real-time PCR assay based on the differential expression of microRNAs and protein-coding genes for molecular classification of formalin-fixed paraffin embedded medulloblastomas

Abstract

Background: Medulloblastoma has recently been found to consist of 4 molecularly and clinically distinct subgroups: WNT, Sonce hedgehog (SHH), Group 3, and Group 4. Deregulated microRNA expression is known to contribute to pathogenesis and has been shown to have diagnostic and prognostic potential in the classification of various cancers.

Methods: Molecular subgrouping and microRNA expression analysis of 44 frozen and 59 formalin-fixed paraffin embedded medulloblastomas from an Indian cohort were carried out by real-time RT-PCR assay.

Results: The differential expression of 9 microRNAs in the 4 molecular subgroups was validated in a set of 101 medulloblastomas. The tumors in the WNT subgroup showed significant (P < .0001) overexpression of miR-193a-3p, miR-224, miR-148a, miR-23b, and miR-365. Reliable classification of medulloblastomas into the 4 molecular subgroups was obtained using a set of 12 protein-coding genes and 9 microRNAs as markers in a real-time RT-PCR assay with an accuracy of 97% as judged by the Prediction Analysis of Microarrays. Age at diagnosis, histology, gender-related incidence, and the relative survival rates of the 4 molecular subgroups in the present Indian cohort were found to be similar to those reported for medulloblastomas from the American and European subcontinent. Non-WNT, non-SHH medulloblastomas underexpressing miR-592 or overexpressing miR-182 were found to have significantly inferior survival rates, indicating utility of these miRNAs as markers for risk stratification.

Conclusions: The microRNA based real-time PCR assay is rapid, simple, inexpensive, and useful for molecular classification and risk stratification of medulloblastomas, in particular formalin-fixed paraffin embedded tissues, wherein the expression profile of protein-coding genes is often less reliable due to RNA fragmentation.

Keywords: Indian cohort, medulloblastoma; miRNA; molecular classification; risk stratification.

Fig. 1.

(A) Heat map showing differential expression of 12 protein-coding genes and 9 miRNAs in the 101 tumor tissues (2 tumors lacking miRNA profile excluded). *indicates the tumor tissues classified primarily based on miRNA expression profile. Subgroup assignment based on PAM analysis using 42 fresh frozen tumor tissues as a training set is indicated above the heat map. (B) The scatter dot plot shows log₂ transformed RQs of the indicated miRNA in the 101 medulloblastomas assigned to the 4 molecular subgroups. The P values given on the top of each scatter indicates the significance of the differential expression of the marker gene in the 4 subgroups as determined by ANOVA tests.

Fig. 2.

The results of PAM analysis showing the subgroup prediction matrix and the predicted test probabilities of the test set based on the expression profile of 12 protein-coding genes and 9 miRNAs.

Fig. 3.

(A) The demographic distribution of the 4 molecular subgroups in the present cohort; (B) subgroup distribution with respect to the age at diagnosis; (C) gender; (D) histological variants. The numbers indicate the number of tumors in each category.

Fig. 4.

Overall survival analysis of (A) 4 molecular subgroups; (B) histological variants; (C) SHH subgroup tumors with and without MYCN overexpression; (D) Group 3 vs Group 4 tumors; (E) non-SHH, non-WNT tumors with or without miR-592 overexpression; (F) non-SHH, non-WNT tumors with or without miR-182 overexpression. P value indicates level of significant difference in the Kaplan–Meier survival curves estimated by the log-rank test.