Single-Cell Sequencing in Pediatric Acute Leukemia: A Systematic Review of Biologic Insights and Translational Potential

Abstract

Jacopo Martelli

Background: Relapse remains the principal cause of treatment failure in pediatric acute leukemia. Clinically validated minimal residual disease (MRD) assays — multiparameter flow cytometry, immunoglobulin/T-cell-receptor rearrangement sequencing and targeted next-generation sequencing panels — are highly sensitive but read out predefined features and do not reconstruct clonal architecture or link a cell’s genotype to its functional state. We systematically reviewed what single-cell sequencing of pediatric acute leukemia has revealed about clonal evolution, chemoresistance, the immune microenvironment and post-therapeutic immune dynamics.

Methods: This review followed PRISMA 2020. PubMed/MEDLINE, the Cochrane Library and Google Scholar were searched to September 2026 for primary studies applying single-cell DNA, RNA, chromatin-accessibility, surface- protein or immune-repertoire sequencing to pediatric acute leukemia samples or pediatric-derived models. Risk of bias in non-randomized studies was assessed with ROBINS-I. Because designs, platforms and outcomes were heterogeneous, findings were synthesized narratively by biologic theme without meta-analysis, following the SWiM guideline.

Results: Of records identified, studies were included (published 2016–2026, 6–159 patients or samples per study, up to ~700,000 cells profiled). Single-cell DNA sequencing traced minor NOTCH1-mutant diagnostic subclones into PTEN- or TP53-mutant relapse clones that bulk sequencing missed. Rare dormant, label-retaining cells and developmental- state-dependent drug sensitivity defined chemo resistant compartments. Remodeling of the bone marrow immune microenvironment identified patients with adverse outcome among those classified as MRD-low, and pre-infusion CAR T-cell type 2 functionality and post-transplant cytotoxic memory CD8+ T-cell subsets were associated with durable remission. Of the seven studies assessed with ROBINS-I, four were at moderate and three at serious overall risk of bias, driven chiefly by small, selected cohorts.

Conclusions: Single-cell sequencing yields mechanistic insight that complements, rather than replaces, clinically validated MRD assays, and nominates candidate biomarkers for risk stratification, treatment selection, cell-therapy manufacturing and post-transplant monitoring. The evidence remains preliminary, adequately powered, prospective and harmonized studies are required before clinical adoption.

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