DNA methylation patterns allow the subclassification of medulloblastoma (MB) into 4 molecular subgroups, which inform treatment and risk-stratification. Whilst microarrays to assign subgroup are suitable for research, their clinical utility is limited by expense, platform-specificity, sample quality requirements and practicality. Here, we aimed to develop a low-cost, array-independent, robust subgrouping assay suitable for routine quality-controlled subclassification, including scant, poor-quality, aged samples, and apply it to the previously unassignable PNET4 trial.
Using a cross-validated classification model, a minimal, multiply-redundant, 17-locus signature was derived to assign subgroup from 220 MBs profiled using Illumina 450k DNA-methylation arrays. We next adapted the MALDI-TOF Mass Spectometry (MassARRAY, Agena Bioscience) iPLEX assay to interrogate DNA methylation following bisulfite treatment. After in vitro validation, the assay was applied to 101 DNA extracts from fresh-frozen, FFPE and nuclear (
95/101 validation samples had high-confidence assignments which recapitulated 450k subgroup calls. Subsequently, high-confidence calls were made for 107/153 PNET4 samples. Notably, a worse survival was observed for standard-risk PNET4 Grp4 patients (EFS 80%; p = 0.01).
MBs can be routinely subgrouped using minimal DNA methylation signatures. The assay is suitable for reliable, robust testing of poor-quality, degraded samples using
Clinical trial identification
Legal entity responsible for the study
Cancer Research UK The Brain Tumour Charity Children's Cancer Run
All authors have declared no conflicts of interest.