Abstract Introduction: Diffuse gliomas are the aggressive primary brain tumor, characterized by poor prognosis and pronounced molecular heterogeneity. Although systemic spread is uncommon due to the blood-brain barrier, tumor cells and nucleic acids can still reach the peripheral circulation. Liquid biopsy, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides a minimally invasive approach for tumor characterization and monitoring in diffuse gliomas. Methods: After validating our CTC detection workflow using spiking experiments with the U87-MG cell line, we analyzed peripheral blood samples from patients undergoing curative resection for diffuse gliomas. Samples were collected prior to surgery in Cell-Free DNA BCT® tubes (Streck, Inc. ). CTCs were identified using the CytoTrack CT11™ (2/C) semi-automated immunofluorescence microscopy platform, with immunostaining for GFAP, vimentin, and CD45, and DAPI nuclear counterstaining. For ctDNA analysis, cfDNA was isolated from 2 ml of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen), and the IDH1 R132H mutation was assessed by digital droplet PCR (Bio-Rad Laboratories, Inc. ). Results: CTCs were detected in 29. 1% of 110 blood samples collected from patients at the time of their first surgery. Among 19 samples from relapsing patients, CTC positivity increased to 54. 5%. Kaplan-Meier survival analysis in a cohort of 67 patients with IDH wildtype glioblastoma and at least two years of follow-up revealed no significant association between CTC status and overall survival. Analysis of cfDNA identified IDH1 R132H mutations in 14% of patients with diffuse gliomas, including cases with both tumor-informed positive and negative status. Conclusion: Our results demonstrate that both CTCs and ctDNA can be reliably detected in patients with diffuse gliomas, underscoring the potential of liquid biopsy to complement traditional tissue-based diagnostics. Detection of IDH1 R132H mutations in plasma— even in tumor-informed negative cases—may indicate intratumoral heterogeneity, compartmentalized tumor biology, or sampling limitations. The lack of survival association may reflect the overall short life expectancy in GBM and/or restricted tumor proliferation outside the CNS. Patient recruitment and data collection remain ongoing. Acknowledgment: This study was supported by European Union - Next Generation EU (LX22NPO5102), Palacky University Olomouc (IGA LF 2025₀06), and SALVAGE - CZ. 02. 01. 01/00/22₀08/0004644. Citation Format: Josef Srovnal, Pavel Stejskal, Ondrej Kalita, Marek Slachta, Lumir Hrabalek, Alona Rehulkova, Monika Vidlarova, Marian Hajduch. Molecular profiling of diffuse gliomas via liquid biopsy: Insights from circulating tumor cells and ctDNA analysis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (7 Suppl): Abstract nr 1070.
Srovnal et al. (Fri,) studied this question.