Emergence of a leukemic clone with a deletion of Tyr260 in CD19. (A) Diagnostic flow cytometry demonstrates loss of surface CD19 during treatment with CD19-targeted immunotherapies. Scatter plots of pretreatment (bone marrow) and postblinatumomab (peripheral blood) lymphoblasts stained with the anti-CD19 antibody FMC63 and costained with antibodies against CD10 (left). Histograms of CD19 and CD10 expression by lymphoblasts and other hematopoietic cells pre- and postblinatumomab treatment (right). (B) Predictive protein structural modeling of wild-type (WT) and ΔY260 CD19. Residues essential for FMC63 binding are shown in blue, and residues surrounding Tyr260 are shown in orange. (C) Schematic representation of inferred evolutionary trajectory of CD19ΔY260. Dark blue ellipse represents a clone that was detectable at diagnosis but disappeared after initial chemotherapy. Table below reflects allele frequency of each clone over time normalized to percentage of cells collected that were malignant (∼90% for clone 3 across time points). (D) Quantification of B-cell lineage transcripts in diagnosis, postblinatumomab, and post–CAR T-cell treatment ALL cells.