Mechanisms of CD19 loss during blinatumomab treatment. (A) Protein domain plot of CD19 mutations identified in posttreatment CD19⁻ relapse samples. (B) Analysis of CD19 locus and CD81 mutations in pre- and posttreatment samples. (C) MAF of CD19 mutations by blast percentage in CD19⁻ relapse samples. (D) Cell surface expression of CD19 (x-axis) in NIH-3T3 cells with MSCV-IRES-RFP (MIR) empty vector (EV), wild-type (WT) CD19, Tyr259fs-1 (c.775 T→TGT), and Tyr259fs-2 CD19 mutations (c.776 A→ATTGGAGATCCC) by flow cytometry (top) and immunofluorescence (bottom). Images were taken at 40× magnification using Nikon C2 confocal on TE2000 ET microscope. Scale bars, 25 μm. CD19, green; RFP, red; DAPI, blue. (E) Protein domain plots for 4 CD19⁻ relapse samples subject to targeted sequencing. Enlarged font represents mutations identified by WES (100× coverage); smaller font represents mutations identified by targeted sequencing of CD19 (20 000× coverage). CPM, count per million; DAPI, 4′,6-diamidino-2-phenylindole; hypo, hypodiploid; RFP, red fluorescent protein.