1. Clinical utility of low-pass whole genome sequencing (LP-WGS) and targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors

We designed a liquid biopsy (LB) platform combining LP-WGS and targeted mutation and gene fusion analysis of cell-free (cf) DNA for diagnosing and monitoring pediatric patients with solid tumors. Sample types included cerebrospinal fluid (CSF) (brain tumors), aqueous humor (retinoblastoma), and blood (sarcomas, germ cell and renal tumors). cfDNA was extracted at diagnosis, during therapy, at remission and/or relapse. WGS libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA and paired-end sequenced (Illumina NextSeq 500) for an average depth of coverage across the genome of 3x. Copy number (CN) profiles were analyzed using the iChorCNA R package and compared with CN abormalities (CNAs) of the primary tumors detected with CytoScanHD or Oncoscan arrays. Selected cases were analyzed for mutations or gene fusions using a custom glioma or cancer predisposition hybrid capture-based panel (Twist Bioscience). Mutations identified in LBs were compared with those in the primary or metastatic tumors with our OncoKids NGS panel. Marked genomic instability in osteosarcoma was readily detectable by LP-WGS. With one exception, CNAs detected by LP-WGS mirrored those of the primary tumors. In one patient with Wilms tumor, CNAs in the LB were distinct from the primary tumor, but consistent with CNAs in two bilateral lung metastases. One patient with osteosarcoma demonstrated an abnormal clone in the LB 12 months off therapy, prior to recurrence in the lung. These examples support a role of LB in early detection of relapse and metastasis. KIAA1549-BRAF fusions were detected in CSF from patients with pilocytic astrocytoma, RB1 mutations and 6p gains were detected in retinoblastoma patients, and an isochromosome 12p was identified in germ cell tumor patients. These data suggest that NGS-based LB assays may be used to monitor pediatric patients with a variety of solid tumors, from diagnosis through treatment and recurrence.