Background: Minimal residual disease (MRD) is a critical prognostic marker in acute lymphoblastic leukemia (ALL). The well studied and used MRD detection methods, multiparametric flow cytometry (MFC) and real-time quantitative polymerase chain reaction (qRT-PCR) for fusion genes and receptor gene rearrangements have significantly improved risk stratification, but have limitations in sensitivity and applicability. Next-generation sequencing (NGS) has emerged as a promising approach for MRD assessment, offering better sensitivity and the ability to track clonal evolution.Objectives: This systematic review evaluates the clinical utility and prognostic value of NGS for MRD detection in ALL, comparing its performance with conventional methods and exploring its potential role in therapeutic guidance.Methods: A comprehensive literature search was conducted across PubMed and Web of Science following PRISMA guidelines. Studies were included if they assessed MRD using NGS in ALL patients and provided data on sensitivity and prognostic value. Comparative analyses with MFC or qRT-PCR were considered. Data on end-of-induction MRD values, event-free survival (EFS), and overall survival (OS) were extracted.Results: Thirteen studies met the inclusion criteria. NGS demonstrated superior sensitivity in detecting MRD-positive cases compared to MFC in patients classified as MRD-negative. Higher correlation was observed in MRD-positive cases than in MRD-negative cases. NGS-based MRD stratification correlated strongly with clinical outcomes, with patients achieving NGS-MRD negativity exhibiting superior EFS and OS rates. Additionally, NGS was highly predictive of relapse following hematopoietic stem cell transplantation and CAR-T cell therapy. The IGH rearrangements as the primary marker in NGS panels has demonstrated good prognostic value in B-ALL.Conclusion: NGS represents a transformative tool for MRD monitoring in ALL, offering enhanced sensitivity and prognostic accuracy. Challenges such as high costs, complex bioinformatics analysis and the need for standardization remain. While its integration into clinical practice holds significant promise, further research is needed to establish standardized protocols, cost-effectiveness, and its optimal role in treatment decision-making. The combination of NGS with MFC may provide complementary advantages.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and genomic profiling has become increasingly vital for risk stratification. We conducted a retrospective single-center study including 96 newly diagnosed pediatric patients with ALL and mixed phenotype acute leukemia to characterize the genomic landscape using conventional and molecular cytogenetics, multiplex ligation-dependent probe amplification and targeted next-generation sequencing and to evaluate associations with treatment response and survival. Mutation co-occurrence analysis revealed a NRAS/KRAS/JAK cluster and an ETV6::RUNX1 cluster characterized by relative mutational exclusivity, with additional associations between NOTCH1–CDK4 and CDK4–CCND3 alterations. DelCDKN2A/CDKN2B alterations showed a trend toward higher day-15 minimal residual disease (MRD) levels and inferior 2-year event free survival rate (EFS). DelIKZF1-positive cases showed lower 2-year EFS. In contrast, ETV6::RUNX1 and high hyperdiploidy were associated with favorable early response and EFS. DelPAX5 did not independently influence outcome. ZNF384 positive cases showed higher early MRD levels despite excellent survival outcomes. NRAS/KRAS mutations were significantly associated with higher positive day-15 MRD (Wilcoxon, p = 0.0067) and remained independently associated after adjustment for white blood cell count and cytogenetic subgroup. Intermediate risk (IR) and high-risk groups showed comparable 2-year EFS, indicating limited discrimination by conventional risk stratification. The IR group displayed a heterogeneous genomic profile, with NRAS/KRAS, Ph-like mutations and delCDKN2A/CDKN2B among the most frequent alterations. These observations highlight the potential of integrated genomic profiling to refine risk stratification, particularly by identifying clinically relevant subgroups within the IR category.