Enhancing Molecular Classifying Accuracy of Pediatric CNS Tumors: A Dual-Classifier Approach Using DNA Methylation Profiling

Background Accurate classification of pediatric central nervous system (CNS) tumors is critical for optimal treatment yet remains challenging due to the limitations of traditional histopathological methods. DNA methylation profiling has gained attention as a promising tool for the molecular classification of CNS tumors. However, despite its potential clinical value, methylation classifiers remain limited to the research settings. This study aims to assess the use of two DNA methylation-based classifiers for CNS tumor diagnostics, with the eventual goal of integrating them into clinical practice and the impact of technical factors such as fixation methods, DNA quantity, and array choice, while exploring the utility of visualization tools (UMAP/t-SNE) and the integration of molecular data for resolving diagnostic ambiguities.

We analyzed 96 pediatric pathology tissue samples, including 75 CNS tumors, 10 with CNS non-tumoral lesions and 11 with non-CNS tumors, performing 130 methylation analyses. DNA from both formalin-fixed paraffin-embedded (FFPE) and fresh frozen (FF) tissues were used for methylation profiling using the Illumina MethylationEPIC V1 and V2 arrays. The performance of two DNA methylation-based classifiers (Heidelberg and NIH) was evaluated by comparing the classification results with histopathological diagnoses. Technical variables that may affect quality such as DNA quantity, extraction method, and sample fixation were also investigated.

Both classifiers demonstrated an 88% concordance with histopathological diagnoses in CNS tumors. Methylation profiling refined the histological diagnoses in 54.66% of cases and contributed to molecular subtyping in 52% of CNS tumor cases. The analysis in a small percentage of cases (5.33%) exhibited conflicting diagnoses, emphasizing the need for cautious interpretation and re-evaluation of the cases of uncertainty. Interestingly, both classifiers also identified CNS non-tumor tissues from tumor cases, although they misclassified some normal tissues and malformations as CNS tumors. Technical factors, including DNA quantity and sample fixation, had minimal impact on classifier performance.

This study highlights the potential of DNA methylation profiling as a complementary diagnostic tool in pediatric CNS tumor classification, paving the way for its integration into routine clinical practice. To the best of our knowledge, this is the first publication comparing two DNA methylation classifiers in a pediatric CNS tumor cohort. While the classifiers show promise for improving diagnostic accuracy, especially in complex or undiagnosed cases, they should be used as a complementary tool to histopathologic classification. Further research is needed to validate their integration into clinical practice, including refining technical protocols, addressing limitations, and evaluating long-term clinical outcomes. Competing Interest Statement The authors have declared no competing interest. Funding Statement This work was funded by D.B. internal PI budget and W.H. internal PI budget provided by Sidra Medicine research branch. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All human samples used in this study were collected under approval from the Sidra Medicine Institutional Review Board (IRB Protocol #150900). For samples obtained without individual consent, the IRB approved their use in research in an anonymized format for assay development. These samples contain no personally identifiable information and are limited to metadata such as age, gender, and tumor type. No clinical history or survival data were used, and the samples cannot be traced back to individual patients. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors Abbreviations - DNA - Deoxyribonucleic acid - FFPE - Formalin-Fixed Paraffin-Embedded - FF - Fresh frozen - MP - Methylation profiling