Development and clinical validation of molecular subgrouping in medulloblastoma by targeted methylation sequencing

Abstract

BackgroundThe WHO classification of CNS tumors confers promising prognostic value to the molecular classification of medulloblastoma (MB). Next-generation sequencing (NGS) has been the primary method employed for molecular classification through transcriptomic, genomic, or methylation profiling. However, due to cost and infrastructural needs, particularly in developing countries, we propose a relatively simple, rapid, and economical Sanger sequencing-based targeted methylation sequencing method for MB classification and prognostication. MethodsEleven epigenetic targets were amplified using optimized primers and bisulfite-converted DNA for Sanger sequencing. Chromas software was used for low-quality data trimming and NCBIs Needleman Wunsch alignment tool was used for sequence alignment to reference. The developed method was applied to tissues from twelve cases of medulloblastoma. ResultsSuccessful interpretation of methylation status in ten out of eleven targets was achieved which was sufficient for classification according to the latest WHO classification of Medulloblastoma tumors. Twelve medulloblastoma cases were classified into WNT (n=2), Group 3 (n=5), and Group 4 (n=5). ConclusionThe developed Sanger sequencing method is a cost-effective, in-house solution that can be used for molecular subgrouping of medulloblastoma. It offers an alternative to NGS, can be done on a case-to-case basis, and does not require high-end infrastructure, sample pooling, or extensive bioinformatics knowledge. Impact statementMolecular classification is imperative for determining the prognosis of medulloblastoma and is recommended by WHO. However, NGS proves to be an expensive option in developing countries. This study has come up with an affordable targeted methylation Sanger sequencing method requiring minimal bioinformatic skills, by utilizing epigenetic targets, for prognostication and risk stratification in medulloblastoma patients. The molecular subgroups of all recruited cases were successfully determined according to WHO classification which is crucial information that, when combined with clinical findings, will enable the clinicians to determine effective treatment strategies.