Genomic fusion breakpoints for DNA-based measurable residual disease monitoring in pediatric acute lymphoblastic leukemia.

Measurable residual disease (MRD) is a key prognostic factor in pediatric acute lymphoblastic leukemia (ALL), but current gold-standard methods based on immunoglobulin/T-cell receptor (IG/TCR) rearrangements are complex and not informative in a subset of patients. Genomic breakpoints of oncogenic fusions (GFBs) are stable, biologically grounded markers that may overcome these limitations and improve MRD assessment. In a cohort of 403 patients with known or suspected fusions, a short-read, capture-based next-generation sequencing strategy coupled with an open-source pipeline identified patient-specific GFBs in 97% of cases. GFB-based MRD assays implemented by qPCR or ddPCR showed very high specificity and allowed lower detection thresholds, resulting in improved sensitivity relative to IG/TCR-based assays. Longitudinal monitoring in 104 patients across multiple pediatric ALL fusion subtypes demonstrated excellent overall concordance between methods in non-BCR::ABL ALL, while revealing fusion-dependent differences. GFB-based MRD was particularly informative in ETV6::RUNX1- and MEF2D-rearranged ALL, where IG/TCR tracking is frequently suboptimal or unreliable due to ongoing rearrangements or the absence of VDJ recombination, respectively. Altogether, this large-scale study establishes GFBs as robust, clinically implementable MRD markers and supports their integration into clinical strategies, provided that subtype-specific biology is considered when selecting markers and interpreting results.