Uncovering the potential of circulating tumor DNA for pediatric precision oncology, the INFORM experience

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 prospective multicenter pediatric precision oncology program INFORM, we collected liquid biopsies from 130 pediatric patients and optimized cfDNA 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 biopsys 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 for the molecular alteration-informed selection and implementation of these complementary approaches within molecular tumor boards, with the aim of optimizing clinical management strategies for high-risk pediatric cancer patients. SignificanceSystematic 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.