Development of Cell-free DNA-based Liquid Biopsy for Early Diagnosis of Brain Injury

Introduction: Detection of tissue-specific cell injury using methylation patterns on cell-free DNA (cfDNA) has formed the basis of liquid biopsy for oncologic disease as well as other pathologies. We aimed to test the ability of cfDNA-based liquid biopsy to support early brain injury diagnosis.

Methods: Digital droplet PCR (ddPCR) assays were designed to amplify hypomethylated genomic loci in brain neurons. Their robustness and sensitivity were evaluated using solutions of brain tissue DNA spiked into leukocyte DNA. The best-performing assay was applied to plasma samples collected from patients with traumatic brain injury (TBI) or intracranial hemorrhage (ICH) within 24 hours of injury. DdPCR readout was correlated with injury features. Targeted proteomics was performed in the same plasma samples to test for association with biomarkers of neuroglial injury.

Results: Five methylation-based ddPCR assay designs were developed and tested. The most promising assay detected brain tissue DNA at spike-in concentrations as low as 0.5%, and spike-in concentration was correlated with ddPCR readout (r=0.984). Plasma was collected from 20 TBI/ICH patients (55% male, 63.4±18.0 yrs). Brain neuron-derived cfDNA was detected in N=12/20 injured patients and N=2/17 non-injured controls (AUC=0.732). Median concentration of brain neuron-derived cfDNA was higher in patients with severe (GCS:3-8) versus mild (GCS:13-15) injury (5.30 vs. 0.00 hGE/mL, P=0.038). Among injured patients, brain neuron-derived cfDNA demonstrated moderate ability to distinguish those who suffered mortality (AUC=0.764). Brain neuron-derived cfDNA concentration was moderately correlated with GFAP (r=0.359), UCHL1 (r=0.427), and S100B (r=0.491).

Conclusion : Our findings support the potential of brain neuron-derived cfDNA to serve as a blood-based biomarker of brain injury. Further optimization to improve its diagnostic abilities is needed.