Validation of an Adapted NGS-Based Clonality Assay for MRD Monitoring in B-ALL Using Archived and Follow-Up Samples.

Background: Measurable residual disease (MRD) monitoring is a critical component of risk stratification and therapeutic decision-making in B-cell acute lymphoblastic leukemia (B-ALL). Multiparameter flow cytometry (MFC) is widely used, but its sensitivity is limited to 10-4-10-5. Next-generation sequencing (NGS) platforms offer greater sensitivity (up to 10-6) but are often limited by cost, infrastructure requirements, and reliance on FFPE tissue. We evaluated the performance of a commercially available NGS-based assay, the Oncomine B-cell Clonality panel, adapted at our center for MRD monitoring using diverse sample types. Methods: In this retrospective study, 32 B-ALL patients were analyzed. Diagnostic clonotypes were identified from archival stained bone marrow aspirate smears in all cases, and FFPE bone marrow tissue in five. Longitudinal MRD monitoring was performed on bone marrow aspirates collected in EDTA. Serial dilution experiments (10-2-10-6) were conducted to assess sensitivity; reproducibility was tested in triplicates across runs. MRD results from NGS were compared to MFC, and statistical agreement was evaluated using Spearman's correlation and Cohen's kappa. Results: Baseline clonotypes were detected in 29/32 patients (90.6%). The assay demonstrated a limit of detection of 10-6 and high reproducibility (R 2 = 0.991). NGS and MFC showed 86.5% concordance; NGS detected MRD earlier than MFC in eight cases (44-124 days). Sensitivity and specificity of NGS relative to MFC were 100% and 82.98%, respectively. Clonal detection succeeded in both FFPE and stained slides. Conclusion: This adapted NGS assay offers a sensitive, reproducible, and sample-flexible MRD monitoring tool for B-ALL, with potential for broader clinical application. Trial Registration: The authors have confirmed clinical trial registration is not needed for this submission.