15‐color highly sensitive flow cytometry assay for post anti‐CD19 targeted therapy (anti‐CD19‐CAR‐T and blinatumomab) measurable residual disease assessment in B‐lymphoblastic leukemia/lymphoma: Real‐world applicability and challenges

Abstract

AbstractObjectivesMeasurable residual disease (MRD) is the most relevant predictor of disease‐free survival in B‐cell acute lymphoblastic leukemia (B‐ALL). We aimed to establish a highly sensitive flow cytometry (MFC)‐based B‐ALL‐MRD (BMRD) assay for patients receiving anti‐CD19 immunotherapy with an alternate gating approach and to document the prevalence and immunophenotype of recurrently occurring low‐level mimics and confounding populations.MethodsWe standardized a 15‐color highly‐sensitive BMRD assay with an alternate CD19‐free gating approach. The study included 137 MRD samples from 43 relapsed/refractory B‐ALL patients considered for anti‐CD19 immunotherapy.ResultsThe 15‐color BMRD assay with CD22/CD24/CD81/CD33‐based gating approach was routinely applicable in 137 BM samples and could achieve a sensitivity of 0.0005%. MRD was detected in 29.9% (41/137) samples with 31.7% (13/41) of them showing <.01% MRD. Recurrently occurring low‐level cells that showed immunophenotypic overlap with leukemic B‐blasts included: (a) CD19+CD10+CD34+CD22+CD24+CD81+CD123+CD304+ plasmacytoid dendritic cells, (b) CD73bright/CD304bright/CD81bright mesenchymal stromal/stem cells (CD10+) and endothelial cells (CD34+CD24+), (c) CD22dim/CD34+/CD38dim/CD81dim/CD19−/CD10−/CD24− early lymphoid progenitor/precursor type‐1 cells (ELP‐1) and (d) CD22+/CD34+/CD10heterogeneous/CD38moderate/CD81moderate/CD19−/CD24− stage‐0 B‐cell precursors or ELP‐2 cells.ConclusionsWe standardized a highly sensitive 15‐color BMRD assay with a non‐CD19‐based gating strategy for patients receiving anti‐CD19 immunotherapy. We also described the immunophenotypes of recurrently occurring low‐level populations that can be misinterpreted as MRD in real‐world practice.