Cell-Free DNA Extracted from CSF for the Molecular Diagnosis of Pediatric Embryonal Brain Tumors

Abstract

Background: Liquid biopsies are revolutionary tools used to detect tumor-specific genetic alterations in body fluids, including the use of cell-free DNA (cfDNA) for molecular diagnosis in cancer patients. In brain tumors, cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. Methods: The CSF cfDNA of pediatric patients with medulloblastoma (n = 18), ATRT (n = 3), ETMR (n = 1), CNS NB FOXR2 (n = 2) and pediatric EBT NOS (n = 1) (mean cfDNA concentration 48 ng/mL; range 4–442 ng/mL) and matched tumor genomic DNA were sequenced by WES and/or a targeted sequencing approach to determine single-nucleotide variations (SNVs) and copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was also used for nucleosome footprinting in CSF cfDNA. Results: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n = 15), a mean of 83 (range 1–160) shared SNVs were observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1–62) or CSF-specific SNVs (mean 5; range 0–25) were also observed, suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. Conclusion: CSF cfDNA sequencing yielded informative results in 60% (15/25) of all cases, with informative results in 83% (15/18) of all cases analyzed by WES. These results pave the way for the implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.

Diagnosis of pediatric central nervous system tumors using methylation profiling of cfDNA from cerebrospinal fluid

“…The signi cance of this is highlighted by the wide variation in published collection protocols, encompassing collection through lumbar puncture, ventricular drainage, or mixed cohorts, with volumes ranging from 200µl to 10 ml. While centrifugation is commonly included in most protocols, there are studies that omit this step 30,31,[33][34][35]37,45 .…”

“…Published cfDNA studies on CSF have mostly focused on tumor follow-up rather than classi cation. For example, cfDNA is used for mutation detection where tumors are detected based on the previously de ned patient/tumor speci c mutations [30][31][32][33][34][35] . Since mutations frequently fail to differentiate between tumor subtypes, Li and colleagues instead used whole genome bisul te sequencing (WGBS) based DNA methylation pro ling and hydroxymethylation pro ling through anti-CMS immunoprecipitation sequencing to subtype and monitor medulloblastoma tumors 36 .…”