BACKGROUND: The use of liquid biopsy is of potential high importance for children with high grade (HGG) and diffuse midline gliomas (DMG), particularly where surgical procedures are limited, and invasive biopsy sampling not without risk. To date, however, the evidence that detection of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) could provide useful information for these patients has been limited, or contradictory. METHODS: We optimized droplet digital PCR (ddPCR) assays for the detection of common somatic mutations observed in pediatric HGG/DMG, and applied them to liquid biopsies from plasma, serum, cerebrospinal fluid (CSF), and cystic fluid collected from 32 patients. RESULTS: Although detectable in all biomaterial types, ctDNA presented at significantly higher levels in CSF compared to plasma and/or serum. When applied to a cohort of 127 plasma specimens from 41 patients collected from 2011 to 2018 as part of a randomized clinical trial in pediatric non-brainstem HGG/DMG, ctDNA profiling by ddPCR was of limited use due to the small volumes (mean = 0.49 mL) available. In anecdotal cases where sufficient material was available, cfDNA concentration correlated with disease progression in two examples each of poor response in H3F3A_K27M-mutant DMG, and longer survival times in hemispheric BRAF_V600E-mutant cases. CONCLUSION: Tumor-specific DNA alterations are more readily detected in CSF than plasma. Although we demonstrate the potential of the approach to assessing tumor burden, our results highlight the necessity for adequate sample collection and approach to improve detection if plasma samples are to be used.

Children and Young People's Unit Royal Marsden Hospital NHS Trust Sutton UK

Department of Haematology and Oncology UCL Great Ormond Street Institute for Child Health London UK

Department of Onco haematology Cell and Gene Therapy Bambino Gesù Children's Hospital IRCCS Rome Italy

Department of Paediatric Oncology Great North Children's Hospital Newcastle University Center for Cancer Newcastle upon Tyne UK

Department of Pediatric Oncology University Hospital Brno Children's Hospital Brno Czechia

Department of Pediatrics Institute of Clinical Sciences Queen Silvia Children's Hospital University of Gothenburg Gothenburg Sweden

Department of Radiology Nottingham University Hospital Trust Nottingham University Hospital Trust Nottingham UK

Division of Clinical Studies The Institute of Cancer Research London UK

Division of Molecular Pathology Institute of Cancer Research London UK

Medical Physics and Clinical Engineering Nottingham University Hospital Trust Nottingham University Hospital Trust Nottingham UK

Molecular Diagnostics Royal Marsden Hospital NHS Trust Sutton UK

Pediatric and Adolescent Oncology and INSERM Unit U981 Team Genomics and Oncogenesis of Pediatric Brain Tumors Gustave Roussy and Paris Saclay University Villejuif France

Pediatric Onco Hematology Department University Hospital of Strasbourg Strasbourg France

UMR CNRS 7021 Laboratory Bioimaging and Pathologies Tumoral Signaling and Therapeutic Targets team Faculty of Pharmacy Illkirch France

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