Accurate classification of ependymomas and medulloblastomas using Raman spectroscopy and pilot transcriptomic profiling.

Raman spectroscopy enabled accurate discrimination of posterior fossa ependymomas and medulloblastomas in both frozen and formalin-fixed, paraffin-embedded (FFPE) specimens in this retrospective study. We acquired Raman spectra (532 nm excitation) from frozen and FFPE tissues to evaluate a principal component analysis-support vector machine classifier by using fivefold cross-validation. We also performed a pilot spatial transcriptomics analysis on three FFPE sections by using the 10× Genomics Xenium In Situ v2 FFPE workflow. In total, 34 specimens (21 frozen and 13 FFPE) were analyzed, and the classification models achieved >90% accuracy in distinguishing ependymomas from medulloblastomas under spectrum-level fivefold cross-validation, suggesting discriminative biochemical differences, whereas patient-level performance requires further validation in larger cohorts. Ependymomas had higher lipid-associated Raman bands (1084, 1128, and 1654 cm-1), whereas medulloblastomas exhibited higher deoxyhemoglobin-related Raman bands (1356, 1548, and 1604 cm-1). Compared with normal controls, tumor tissues had increased carotenoid-related Raman bands and reduced lipid-associated Raman bands. The observed spectral differences are consistent with differences in lipid-associated composition and the heme/oxygenation-related tissue context, and pilot transcriptomic profiling provided qualitative biological context related to lipid metabolism and angiogenesis. These findings support Raman spectroscopy as a label-free spectroscopic technique that may complement conventional diagnostics and aid surgical and therapeutic decision-making. Larger, prospective studies are warranted to further evaluate the clinical generalizability and intraoperative translation of our results.