Circulating tumor DNA precision oncology enables effective and sensitive molecular diagnostics and actionable target...

Pediatric solid high-risk malignancies mostly lack established molecular biomarkers for early detection, minimal residual disease assessment, or treatment monitoring. Challenges include small patient numbers, limited sample volumes, low tumor mutational burden, and few recurrent alterations.

Within the multicenter pediatric precision oncology program INFORM, we prospectively collected liquid biopsies from 130 pediatric patients and optimized cell-free DNA isolation and analysis. Whole-genome, whole-exome, and targeted panel sequencing were performed using liquid biopsy-adapted protocols.

Integrating tissue-derived molecular profiles and orthogonal validation revealed that low-coverage whole-genome sequencing reliably detects circulating tumor DNA. An in silico ctDNA estimation score, combining fragment length and genome segment alterations, improved sensitivity and specificity to 95%, enabling plasma-based tumor detection in 93% of patients. Whole-exome and panel sequencing effectively identified clinically relevant, potentially druggable molecular targets. However, their utility varied substantially across different tumor entities, underscoring the need for entity-specific considerations in the interpretation and application of these methodologies. In-depth analyses demonstrated liquid biopsy's potential to track tumor evolution, identifying common tumor ancestors and refining patient stratification.

This study advances liquid biopsy methodologies in pediatric oncology and provides a rationale that, as SNVs are more sensitively captured by panel sequencing and WES, while CNVs are better represented by lcWGS and WES. The underlying tumor genomic profile should guide the selection of liquid biopsy assays to optimize clinical decision-making. Systematic liquid biopsy analyses within the pediatric precision oncology INFORM registry enabled a real-world, multicenter comparison of sequencing approaches across high-risk malignancies. By optimizing preanalytical and bioinformatic tools for pediatric settings, we improved plasma-based cancer detection, molecular tumor characterization, and identification of targetable alterations, laying the groundwork for integration into personalized medicine programs and clinical trials.