Highly sensitive single tube B-lymphoblastic leukemia/lymphoma minimal/measurable residual disease test robust to surface antigen directed therapy.

BACKGROUND:Measurement of minimal/measurable residual disease (MRD) in B-lymphoblastic leukemia/lymphoma (B-ALL) has become a routine clinical evaluation tool and remains the strongest predictor of treatment outcome. In recent years, new targeted anti-CD19 and anti-CD22 antibody-based and cellular therapies have revolutionized the treatment of the high-risk B-ALL. The new treatments raise challenges for diagnostic flow cytometry, which relies on the presence of specific surface antigens to identify the population of interest. So far, reported flow cytometry-based assays are developed to either achieve a deeper MRD level or to accommodate the loss of surface antigens post-target therapies, but not both. METHODS:We developed a single tube flow cytometry assay (14-color-16-parameters). The method was validated using 94 clinical samples as well as spike-in and replicate experiments. RESULTS:The assay was well suited for monitoring response to targeted therapies and reached a sensitivity below 10-5 with acceptable precision (coefficient of variation < 20%), accuracy, and interobserver variability (κ = 1). CONCLUSIONS:The assay allows for sensitive disease detection of B-ALL MRD independent of CD19 and CD22 expression and allows uniform analysis of samples regardless of anti-CD19 and CD22 therapy.