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Pediatric central nervous system (CNS) tumors carry high morbidity and mortality. The advent of targeted agents is aiming to change that through individualized, less invasive, and real-time molecular profiling. We successfully developed a cerebrospinal fluid (CSF) liquid biopsy (LB) platform combining low-pass whole genome sequencing (WGS) and targeted mutation and gene fusion analysis of cell-free (cf) DNA. WGS libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of input cfDNA and paired-end sequenced (Illumina NextSeq 500) for an average 0.5x coverage across the genome. Copy number (CN) profiles were analyzed using the ichorCNA R package and compared with CN abnormalities of the primary tumors detected with CytoScanHD or Oncoscan arrays. Selected cases were analyzed for mutations or gene fusions using a custom glioma hybrid capture-based panel (Twist Bioscience) sequenced to 500-1000x across targeted regions. Mutations identified in LBs were compared with primary tumor OncoKids NGS panel results. CSF LBs from 44 tumor cases and 15 non-tumor controls were analyzed. Sixteen primary tumors had copy number changes, 10 had cfDNA detected in the LB across various primary tumor diagnoses (AT/RT, diffuse midline glioma, ependymoma, high-grade glioma, medulloblastoma, pilocytic astrocytoma). Four primary tumors had sequence alterations from the custom panel, two with detectable cfDNA (diffuse midline glioma H3K27M variant allele frequency (VAF) 49% and low-grade glioma BRAFV600E VAF 1.7%). Three patients had TP53 variants detected, all ultimately germline. Controls were negative across WGS and hybrid gene capture. Tumor DNA is detectable by both WGS and hybrid gene capture. Various histologic subtypes are detectable including embryonal and glioneuronal. CSF seems more enriched for cfDNA in high grade versus low grade tumors. These data suggest that NGS-based LB assays may be used to monitor pediatric patients with a variety of CNS tumors, from diagnosis through treatment and recurrence.

An 11 year old boy presented with a three month history of progressive bilateral lower extremity weakness associated with recent intermittent incontinence. Spine MRI showed a right-sided T11-T12 paraspinal mass extending through the neural foramina into the epidural space and causing severe spinal cord compression. Skin showed a large macular lightly-hyperpigmented café au lait spot with irregular borders along the T10-T12 dermatome extending from the spine to approximately the anterior-axillary line. He was suspected to have segmental neurofibromatosis type 1 (NF1) as no additional clinical findings, radiographic features or family history of NF1 were identified. Patient underwent T10-12 laminectomy for resection of the epidural tumor component. Post-operative MRI showed resolution of the mass effect on the thecal sac and cord, with expected tumor residual lateral to the neural foramen. His residual spinal tumor and mild scoliosis remained stable over the two years of follow up to date. Pathological and molecular analysis of the resected tumor revealed a neurofibroma harboring an activating KRAS c.35G>A, p.Gly12Asp (KRAS-G12D) pathogenic variant at 27% variant allele frequency. Melanocytes cultured from two hyperpigmented skin biopsies showed the same KRAS-G12D pathogenic variant. This KRAS-G12D pathogenic variant was not found in leukocytes, indicating a post-zygotic origin. No NF1 pathogenic variant was identified in tumor tissue, melanocytes or leukocytes. The clinical findings were consistent with a mosaic KRASopathy due to a post zygotic KRAS-G12D pathogenic variant. The presence of the KRAS variant in the spinal neurofibroma and overlaying café au lait spot without an NF1 etiology in associated tissues demonstrates overlapping variability of presentations of RAS-MAPK pathway disorders. This case highlights the need for full clinical and genetic evaluation of patients presenting with segmental neurocutaneous disorders.