Next-generation sequencing as a powerful tool for measurable residual disease assessment in pediatrics: Identifying time points of true remission Free

Next-generation sequencing (NGS) is a sensitive tool for measurable residual disease (MRD) detection, particularly in acute lymphoblastic leukemia (ALL). NGS for IG/TR rearrangements outperforms multiparameter flow cytometry (MFC) and qPCR in identifying patient-specific B-cell receptor and T-cell receptor gene rearrangements. In pediatric ALL, MRD status at treatment milestones such as end of induction (EOI) or consolidation (EOC) is highly prognostic and informs therapy decisions. The most recent pediatric frontline trial AALL1731 incorporated NGS at EOI to enhance MRD-based risk stratification, but data on NGS-MRD kinetics beyond EOI remain limited. It is unknown whether pediatric patients with delayed MRD clearance beyond EOI by NGS—despite MFC negativity— are at a higher risk for relapse and require altered management. This study examines the prognostic relevance of ClonoSEQ NGS-MRD at timepoints beyond EOI for frontline pediatric ALL patients.

Methods We conducted a retrospective analysis (2022-2025) of pediatric patients diagnosed with either B-cell or T-cell ALL who had at least one paired MRD assessment using ClonoSEQ NGS and MFC. NGS-MRD specimens were obtained from bone marrow until NGS-MRD negativity was achieved, then peripheral blood was used for ongoing monitoring at the start of delayed intensification and at the start and end of maintenance therapy. Collected variables included disease subtype, patient demographics, treatment, bone marrow and blood MRD results, and survival outcomes. MFC was used as a comparator assay, with a sensitivity threshold of 1 × 10⁻⁴, while NGS provided enhanced sensitivity at 1 × 10⁻⁶ in the bone marrow.

Results ClonoSEQ NGS was used to evaluate 94 specimens (bone marrow or peripheral blood) from 32 patients with newly diagnosed ALL treated with standard of care frontline protocols. The median age was 10 years (range 2-18), and median follow-up time 18 months (range 1-40). The majority of patients (91%, n=29) had B-ALL, and 66% (n=21) were high-risk based on NCI Rome criteria. Cytogenetic and molecular data were available in 32 patients. Of these, 5 had complex cytogenetics, 10 were diploid, the most common mutation was NRAS (n=4) and 3 had the ETV6-RUNX1 fusion.

Of the 94 specimens, NGS results were compared with MFC data in 60 specimens with paired testing. Discordance between the two modalities was observed in 41% of specimens. Out of 24 MFC negative patients at EOI, 14 were NGS-positive. The median MRD burden at EOI was 8 copies per million cells (range: 1-363445). At the EOC, 15 samples were MFC negative, 8 were NGS-positive.

For these patients quantitative NGS-MRD values showed a progressive decline throughout treatment, and all but 1 ultimately achieved NGS-MRD negativity (2 after immunotherapy alone, 5 after immunotherapy and high dose chemotherapy, 1 after high dose chemotherapy alone). The median time to NGS-MRD clearance was 3 months for all patients. One patient had primary refractory disease with persistent MRD by both NGS and MFC. All T-ALL patients cleared NGS by EOC. Additionally, one T-ALL patient had re-emergence of clones by NGS only in maintenance after several treatment disruptions and remains under close surveillance. No other re-emergence or relapses of MRD have been observed in any patient who achieved MRD-negative remission by either modality. Preliminary survival analysis suggests that patients who became MRD-negative by both assays had a relapse-free survival rate of 100% at current follow-up.

Conclusions ClonoSEQ-based NGS is a highly sensitive tool for leukemia detection from bone marrow or peripheral blood. However, longitudinal data are needed to clarify its role in guiding treatment decisions in pediatrics. In our cohort, NGS detected MRD in cases missed by MFC, particularly at early time points. Persistent NGS positivity beyond consolidation was rare and typically cleared following anthracycline-containing cycles or immunotherapy, underscoring the relevance of these two cycles. T-ALL frequently showed delayed clearance until end of consolidation, but some B-ALL cases also exhibited prolonged NGS positivity without relapse. While our findings highlight the added sensitivity and timing utility of NGS-MRD, the limited sample size precludes conclusions about survival impact or correlation with genetic alterations. Larger studies are needed to validate these trends and refine risk stratification.