Limitations of DNA Methylation Profiling in High-Grade Gliomas: Case Series

<b>Background and Clinical Significance</b>: DNA methylation profiling has revolutionized the classification of central nervous system (CNS) tumors, providing insights into tumor prognosis, recurrence, and personalized treatments. However, a significant challenge remains in classifying rare or molecularly undefined high-grade gliomas (HGGs) that fail to match existing methylation reference classes. This study evaluates the clinical, histopathological, and molecular characteristics of three unclassifiable cases through a retrospective analysis. Methylation profiling was performed by the National Institute of Health based on the 11b6 and 12b6 of the Heidelberg classifier, as well as the National Cancer Institute/Bethesda classifier. The cases were evaluated for histopathological features, molecular markers, and clinical outcomes. <b>Case Presentation</b>: We present three adult patients with histologically confirmed HGGs that were unclassifiable by standard DNA methylation profiling. All patients presented with diverse clinical and radiographic findings. Histopathological examination confirmed high-grade glial neoplasms in each case. However, methylation profiling failed to yield clear matches for any known class. Instead, profiling suggested indeterminate IDH-wildtype neoplasms with aggressive clinical courses. Following treatment, one patient experienced disease progression and died, while the other two remained without evidence of recurrence at follow-up. <b>Conclusions</b>: These cases underscore the persistent diagnostic challenges posed by CNS tumors that are unclassifiable by current DNA methylation, highlighting the urgent need for expanded reference datasets. While methylation profiling has transformed the field of tumor diagnostics, its limitations still exist. Enhanced collaboration to broaden diagnostic categories is essential to broaden diagnostic classifiers. Until these tools are refined, integration of clinical, histological, and molecular findings is imperative to optimize patient management, improve classification accuracy, and optimize therapeutic outcomes. Unclassifiable HGGs represent a critical gap in CNS tumor diagnostics. Addressing this requires global collaboration to enrich methylation databases. In the interim, a multimodal diagnostic strategy remains essential for the management of these challenging tumors.