Liquid Biopsy of Plasma and Cerebrospinal Fluid for the Detection of Extracellular Tumor DNA as a Tool for Glioma Diagnosis and Genotyping: A Cross-Sectional Observational Pilot Study

BACKGROUND: Liquid biopsy is a promising method used for analyzing tumor-derived genetic material in plasma and cerebrospinal fluid. These biological fluids are characterized by low concentrations of such material. Pre-amplification is hypothesized to enhance detection sensitivity.

AIM: This study aimed to assess the detectability and content of extracellular tumor genetic material in plasma and cerebrospinal fluid from patients with glioma using droplet digital polymerase chain reaction with and without pre-amplification.

METHODS: The study investigated plasma and cerebrospinal fluid samples from 25 patients newly diagnosed with glioma who were scheduled for partial or total tumor resection. DNA was extracted from 2.5–5 mL of cerebrospinal fluid and 5 mL of plasma. Each sample was examined by droplet digital polymerase chain reaction for IDH1 R132H and TERT promoter (C228T and C250T) mutations. Mutation analysis was performed on both isolated and pre-amplified DNA. The false-positive threshold was determined through experiments that analyzed wild-type samples and no-template controls. Statistical analysis was conducted using the Mann–Whitney and Wilcoxon tests for paired data and Spearman’s correlation test.

RESULTS: The sensitivity and specificity of cerebrospinal fluid liquid biopsy without pre-amplification were 14.3% and 100% for grade 1–3 gliomas, respectively, and 50% and 100% for grade 4 gliomas. After pre-amplification, the values were 57.1% and 50% for grade 1–3 gliomas and 75% and 80% for grade 4 gliomas. Extracellular DNA levels showed a significant correlation with tumor volume, malignancy grade, and contrast enhancement pattern (i.e., none, moderate/heterogeneous, intense, and ring-shaped).

CONCLUSION: Liquid biopsy with DNA pre-amplification demonstrates limited sensitivity and specificity in glioma detection. However, it enables assessment of key tumor characteristics, which may be useful for therapeutic decision-making.