Prognostic significance of clinical, histopathological, and molecular characteristics of medulloblastomas in the prospective HIT2000 multicenter clinical trial cohort

Abstract

This study aimed to prospectively evaluate clinical, histopathological and molecular variables for outcome prediction in medulloblastoma patients. Patients from the HIT2000 cooperative clinical trial were prospectively enrolled based on the availability of sufficient tumor material and complete clinical information. This revealed a cohort of 184 patients (median age 7.6 years), which was randomly split at a 2:1 ratio into a training (n = 127), and a test (n = 57) dataset in order to build and test a risk score for this population. Independent validation was performed in a non-overlapping cohort (n = 83). All samples were subjected to thorough histopathological investigation, CTNNB1 mutation analysis, quantitative PCR, MLPA and FISH analyses for cytogenetic variables, and methylome analysis. By univariable analysis, clinical factors (M-stage), histopathological variables (large cell component, endothelial proliferation, synaptophysin pattern), and molecular features (chromosome 6q status, MYC amplification, subgrouping) were found to be prognostic. Molecular consensus subgrouping (WNT, SHH, Group 3, Group 4) was validated as an independent feature to stratify patients into different risk groups. When comparing methods for the identification of WNT-driven medulloblastoma, this study identified CTNNB1 sequencing and methylation profiling to most reliably identify these patients. After removing patients with particularly favorable (CTNNB1 mutation, extensive nodularity) or unfavorable (MYC amplification) markers, a risk score for the remaining "intermediate molecular risk" population dependent on age, M-stage, pattern of synaptophysin expression, and MYCN copy-number status was identified, with speckled synaptophysin expression indicating worse outcome. Test and independent validation of the score confirmed significant discrimination of patients by risk profile. Methylation subgrouping and CTNNB1 mutation status represent robust tools for the risk stratification of medulloblastoma. A simple clinico-pathological risk score was identified, which was confirmed in a test set and by independent clinical validation.

Fig. 1

Molecular subgrouping of medulloblastoma samples. a Molecular subgroups of medulloblastoma samples for which sufficient material was available (n = 179) as assessed by unsupervised k-means consensus clustering of 450k methylation array data. A large subset of this data (n = 169) was previously presented in [9]. b Associations of molecular subgroups with EFS across all treatment groups and comparison with c the molecular stratification planned for the upcoming European cooperative medulloblastoma trial PNET5 (low risk = M0 and residual tumor <1.5 cm² and CTNNB1 mutation positive; high risk = either M1–M4 or MYC/MYCN amplified or residual tumor >1.5 cm² or anaplastic or large cell histology; standard risk = all remaining cases)

Fig. 2

Comparison of markers for the identification of WNT-driven medulloblastomas. EFS for patients with WNT-subgroup tumors as assessed by a 450k methylation analysis, b CTNNB1 exon 3 sequencing, c β-catenin immunohistochemistry (>5 % positive nuclei), d 6q deletion by 450 k. e Venn diagram for assessment of WNT-subgroup markers and their interrelationship: number of WNT-patients according to 450 k-array subgrouping, exon 3 mutation in CTNNB1, beta-catenin IHC (nuclear accumulation of beta-catenin in >5 % of tumor cells) and 6q deletion (as assessed by 450k)

Fig. 3

Identification and test of a risk score. a Example of speckled synaptophysin positivity in contrast to b diffuse synaptophysin positivity. c EFS in the training cohort in which the risk score was established. d EFS in the test cohort, e OS in the training cohort in which the risk score was established, f OS in the test cohort