Abstract PR017: Advancing a comprehensive pediatric liquid biopsy platform, LBSeq4Kids, using Illumina 5-Base technology

Abstract

LBSeq4Kids is a CAP/CLIA-certified pediatric liquid biopsy (LB) platform developed at the Center for Personalized Medicine at CHLA that combines low-pass whole-genome sequencing (WGS) for the detection of copy number alterations (CNAs) and a targeted sequencing panel for the detection of variants and gene fusions from cell-free DNA (cfDNA) isolated from aqueous humor (AH), cerebrospinal fluid (CSF), and plasma. To date, LBSeq4Kids has been used clinically for diagnosis, particularly in RB and brain tumors, and enables serial sampling for longitudinal monitoring, tracking CNAs and variant allele frequency changes on therapy, assessing for residual molecular disease at end of therapy, and detecting relapse or recurrence. We have also demonstrated feasibility of enzymatic methylation (EMSeq) WGS for methylation analysis for brain tumors using CSF; however, limited cfDNA yield from pediatric LB specimens often precludes comprehensive genomic and epigenomic profiling. Illumina 5-Base technology offers an alternative approach, enabling genome-wide methylation profiling alongside copy number analysis and variant/fusion detection from a single assay. Our objective is to clinically validate this integrated platform to consolidate the current three assays, optimize limited pediatric LB specimens, and enable comprehensive first-line molecular testing. Feasibility was first evaluated using four rhabdoid tumor tissue specimens sequenced with 50ng genomic DNA input. Illumina 5-Base demonstrated complete concordance with matched EMSeq WGS for methylation classification using an in-house neural network model and simultaneously identified CNAs and pathogenic SMARCB1 variants. We subsequently processed 20 AH and CSF-derived cfDNA samples with 1.4-5ng cfDNA input from patients diagnosed with retinoblastoma (RB) and central nervous system (CNS) tumors. Illumina 5-Base demonstrated high concordance with the validated LBSeq4Kids platform, accurately detecting clinically relevant CNAs, pathogenic variants, and methylation signatures across RB and pediatric CNS tumors. Findings included pathogenic RB1 variants and recurrent CNAs in AH from patients with RB, subgroup-defining methylation profiles and characteristic CNAs, including isochromosome 17q, in group 3 and group 4 medulloblastomas and an H3K27M mutation in one high grade glioma. Importantly, in one RB case, 5-Base identified RB1 promoter hypermethylation, an alternative second-hit mechanism responsible for only 10-15% of RB cases and not detected by the current clinically validated LBSeq4Kids platform. These preliminary findings demonstrate the feasibility of comprehensive genomic and epigenomic profiling in low-input cfDNA LB samples using Illumina 5-Base technology. By capturing clinically relevant epigenetic alterations, exemplified by medulloblastoma subgroup classification and RB1 promoter hypermethylation, 5-Base expands upon LBSeq4Kids while consolidating three assays into one with the potential to streamline testing and maximize comprehensive cfDNA molecular analysis in pediatric liquid biopsies.