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

Area of Science:

• Hematology
• Oncology
• Molecular Diagnostics

Background:

• Measurable residual disease (MRD) monitoring is crucial for risk stratification and treatment decisions in B-cell acute lymphoblastic leukemia (B-ALL).
• Multiparameter flow cytometry (MFC) is a standard method but has limited sensitivity (10^-4-10^-5).
• Next-generation sequencing (NGS) offers higher sensitivity (up to 10^-6) but faces challenges with cost, infrastructure, and tissue requirements (FFPE).

Purpose of the Study:

• To evaluate the performance of a commercial NGS assay (Oncomine B-cell Clonality panel) for MRD monitoring in B-ALL.
• To assess the assay's sensitivity, reproducibility, and flexibility across diverse sample types.
• To compare NGS-based MRD monitoring with conventional MFC.

Main Methods:

• Retrospective analysis of 32 B-ALL patients.
• Diagnostic clonotypes identified from bone marrow aspirate smears and FFPE tissue.
• Longitudinal MRD monitoring using bone marrow aspirates in EDTA.
• Serial dilution experiments (10^-2-10^-6) for sensitivity assessment.
• Reproducibility testing in triplicates.
• Comparison of NGS results with MFC using statistical analysis (Spearman's correlation, Cohen's kappa).

Main Results:

• Baseline clonotypes detected in 90.6% of patients.
• Assay demonstrated a limit of detection of 10^-6 and high reproducibility (R^2 = 0.991).
• 86.5% concordance between NGS and MFC; NGS detected MRD earlier in 8 cases.
• NGS showed 100% sensitivity and 82.98% specificity compared to MFC.
• Successful clonal detection in both FFPE and stained slides.

Conclusions:

• The adapted NGS assay is a sensitive, reproducible, and sample-flexible tool for MRD monitoring in B-ALL.
• This approach has potential for broader clinical application in leukemia management.
• NGS offers a significant advancement over MFC for MRD detection in B-ALL.