MRD4U: A path to development for personalized liquid biopsy for children with central nervous system tumors

Liquid biopsy assays using cerebrospinal fluid (CSF) can transform care for children with central nervous system (CNS) tumors by enabling precise monitoring of therapeutic responses and detection of residual or recurrent disease. These assays are typically designed to detect cell-free, circulating tumor DNA (ctDNA) via detection of somatic alterations, although the ability to accurately detect and measure low abundance ctDNA in CSF remains challenging. Initially, our research involved the optimization of next-generation sequencing library preparation from cell-free DNA, evaluating four commercial kits to address the challenges of low-input inherent in CSF-derived cell-free DNA. The selected kit showed a capacity to minimize false positives and efficiently detect somatic variants as low as 5% variant allele frequency using 0.1 ng input of a synthetic cfDNA control, demonstrating its suitability for low-volume CSF samples commonly obtained from pediatric patients. We then applied this optimized library preparation protocol to a cohort of six children with CNS tumors, using a hybridization capture approach (MRD4U) designed to enrich for patient-specific tumor variants known from prior exome sequencing. Using MRD4U, two of the CSF samples tested positive for ctDNA (ctDNA+) although both patients had no evidence of clinical or radiological disease at the time of our assay. Furthermore, one of these ctDNA+ patients exhibited radiographic signs of disease recurrence three months after our MRD4U liquid biopsy, underscoring our assay's ability to detect disease earlier and with greater sensitivity. These results highlight the potential of personalized liquid biopsy assays in improving disease monitoring and potentially altering treatment strategies for children with CNS tumors.