1035 - Plasma and CSF Liquid Biopsy Methylomics in Primary or Recurrent Glioblastoma and Radiation Necrosis

Purpose/Objective(s): Radiation therapy is central to glioblastoma treatment, yet distinguishing post-radiation inflammatory changes, including pseudoprogression and radiation necrosis, from true tumor progression remains challenging. These entities can radiographically mimic true tumor progression, creating diagnostic uncertainty that impacts patient management, trial eligibility, and therapeutic decision-making. In many cases, definitive distinction requires months of serial imaging or invasive procedures, delaying appropriate care. We evaluated the diagnostic utility of methylomic profiling of cell-free DNA (cfDNA) in plasma and cerebrospinal fluid (CSF), hypothesizing that methyl-cfDNA reflects divergent biological mechanisms and inflammatory states.

Materials/Methods:: Biobanked plasma and intraoperative CSF samples were obtained from patients with biopsy-confirmed recurrent GBM (6 CSF and 7 plasma) and GBM-associated radiation necrosis (6 CSF and 5 plasma). An expansion cohort for ongoing analysis includes non-neoplastic controls (2 CSF and 3 plasma), pseudoprogression (3 CSF and 3 plasma), and primary GBM (7 CSF and 8 plasma). For all samples, cell-free DNA was extracted and profiled using enzymatic methylation sequencing (EM-seq). Reads were aligned with bwa-meth and filtered to retain properly paired reads with mapping quality =30 on autosomes, while excluding duplicates and blacklisted regions. Differential methylation and downstream analyses were performed using MethylKit and MethylDackel. Reactome overrepresentation analysis was used to identify significantly enriched biological pathways.

Results: In plasma samples from radiation necrosis and recurrent GBM cases, 912 genomic regions were differentially methylated (p<0.05). Of these, 136 were hypermethylated in radiation necrosis compared with recurrence using a more stringent statistical significance threshold (p<0.01). Pathway analysis of hypermethylated genes in radionecrosis samples identified enrichment of IL-6 signaling, MAP kinase, and neurodevelopmental pathways. At the same significance level, 128 loci were hypomethylated and were enriched in pathways related to neuronal signal transduction and extracellular matrix remodeling. CSF samples demonstrated fewer differentially methylated loci (67, p<0.05). In CSF, ABTB2, LCAT1, FANCC, STS, and TLR7 were significantly hypomethylated in radiation necrosis compared with recurrence (p<0.01). No hypermethylated protein-coding loci met this significance threshold in CSF samples.

Conclusion: Plasma and CSF cfDNA methylation profiling reveals significant differences between recurrent glioblastoma and radiation necrosis. These findings support the potential of liquid biopsy methylomics to augment diagnostic decision-making and reduce uncertainty in distinguishing tumor recurrence from treatment-related injury in GBM.