Nanopore-based DNA methylation profiling for rapid molecular classification of NOS/NEC CNS tumors: multi-institutional evaluation using FFPE archives and frozen tissues

BACKGROUND: DNA methylation profiling has become crucial for accurate classification of CNS tumors, yet Illumina EPIC arrays are restricted by long turnaround times, batching requirements, and limited accessibility in non-specialized centers. Rapid, affordable alternatives are urgently needed, particularly in resource-constrained settings. We aimed to evaluate nanopore sequencing as a real-world diagnostic tool for resolving diagnostically challenging CNS tumors. METHODS: We applied the ROBIN pipeline to 34 consecutive CNS tumors that remained NOS/NEC after routine histopathological and molecular workup. Samples comprised 16 routine FFPE blocks and 18 frozen tissues (9 fresh-frozen, 9 pre-extracted DNA) from three pathology centers. Nanopore sequencing was performed on PromethION R10.4.1 flow cells, targeting definitive methylation-based classification report within 24 h. RESULTS: A confident methylation class was assigned in 28/34 cases (82.4%), including 14/16 (87.5%) methylation calls of FFPE samples with median target coverage of 0.03×. Integration of simultaneously generated CNV profiles rescued an integrated glioblastoma diagnosis in two methylation-unclassifiable cases via 7+/10- signatures. The nanopore-based classifications also achieved 84.6% (11/13) concordance with paired result from Illumina Methylation EPIC arrays. In high-coverage samples, diagnostic-relevant SNVs (BRAF V600E, PTEN, NF1/TP53) were recovered from the same run. SNV detection was strongly coverage-dependent, however: it was not achievable in the low-coverage FFPE and fresh-frozen specimens. CONCLUSIONS: Nanopore sequencing enables rapid, accurate, multidimensional molecular classification of diagnostically intractable CNS tumors using routine FFPE and frozen tissue, achieving high diagnostic resolution in comparison with reference methods while drastically reducing turnaround time and cost, making precision neuropathology feasible beyond quaternary centers.