P. S. Chauhan1, I. Alahi1, J. Yuan2, L. Xu2, P. K. Harris3, C. Y. Chien2, S. Fadera2, A. Stark2, H. Chen2, E. C. Leuthardt4, and A. A. Chaudhuri1; 1Department of Radiation Oncology, Mayo Clinic, Rochester, MN, 2Washington University School of Medicine, St Louis, MO, 3Washington University School of Medicine, St. Louis, MO, 4Department of Neurological Surgery, Washington University School of Medicine, St. Louis, MO
Purpose/Objective(s): Blood-based liquid biopsy tests provide a non-invasive means to obtain genomic and epigenomic data. However, circulating tumor DNA (ctDNA) detection in the plasma of glioma patients is difficult due to the presence of the blood-brain barrier (BBB). Focused ultrasound (FUS)-enabled blood-based liquid biopsy (sonobiopsy) locally disrupts the BBB and enriches for brain tumor-derived biomarkers. Here, we evaluate the enrichment of tumor-associated fragmentomic features with sonobiopsy in patients with glioma. Materials/
Methods: Fourteen patients were enrolled prospectively for the clinical study of sonobiopsy at our institution between April 2022 and February 2024 with median age of 63 years (range 36 -74 years). The sonobiopsy procedure was performed prior to tumor resection, using a nimble FUS device integrated with a clinical neuronavigation system. Blood samples were collected before (5 min pre-FUS) and at different time points post-sonication (5, 10, and 30 min post-FUS). Enzymatic Methyl-seq (EM-Seq) was applied to plasma cell-free DNA (cfDNA) isolated from 14 patients and 10 healthy adults. Tumor tissue obtained from 13 glioma patients was also subjected to EM-Seq and whole genome sequencing. We then quantified cfDNA fragmentomic features such as the ratio of short fragments (< 120bp) to long fragments (140-250 bp), median fragment length, and coefficient of variation (CV) of the fragment length distribution at the transcription start sites (TSSs) of all protein-coding genes. Tumor fraction (TFx) levels based on genome-wide copy number alterations in plasma cfDNA was also estimated using ichorCNA. Results: Among the 14 glioma patients prospectively enrolled onto this study, 12 were glioblastoma patients and two were low-grade glioma patients. We analyzed the cell-free DNA sequencing data from five glioma patients and nine healthy adults. Comparing FUS-induced fragmentomic features between pre-FUS and post-FUS samples, the ratio of short to long cfDNA fragments was significantly higher in post-FUS samples (p < 0.0001). Similarly, the median cfDNA fragment size was significantly shorter in post-FUS samples compared to pre-FUS (p < 0.0001), and post-FUS samples had significantly higher cfDNA fragment size CVs at protein-coding TSSs than pre-FUS (p < 0.0001). Furthermore, TFx levels in plasma cfDNA were consistently higher in post-FUS samples compared to pre-FUS. Conclusion: cfDNA fragmentation patterns in glioma patients strongly suggest that post-FUS cfDNA is more fragmented that pre-FUS, consistent with the enrichment of brain tumor-derived DNA within the plasma. These data support the continued investigation of sonobiopsy and cfDNA fragmentomics for the noninvasive molecular diagnosis of brain cancers.
