Emergence of CD19⁻myeloid phenotype blasts after effective CD19 CAR-T-cell therapy for CD19⁺ MLL-rearranged B-ALL. (A-E) Data from the patient in case 1. (F-H) Data from the patient in case 2. (A) The percentage of CD19-specific CAR-T cells in CD3⁺ T cells (open circles) and the absolute CAR-T-cell count (squares) in blood on the indicated days after CAR-T cell infusion are shown for patient 1. CAR-T cells were identified as viable CD45⁺/CD3⁺/epidermal growth factor receptor-positive events in a lymphoid forward scatter/side scatter gate by flow cytometry, and the absolute count was determined by multiplying the absolute lymphocyte count by the percentage of CAR-T cells in a lymphoid gate. (B) Flow cytometry of peripheral blood demonstrating CD19⁺ lymphoblasts (red) before CAR-T-cell therapy. The blasts expressed a low level of CD33 and were largely CD64⁻, CD14⁻, and CD4⁻ (not shown). Flow plots are gated on mononuclear cells. (C) The abnormal blasts (*) before CAR-T-cell therapy were morphologically distinct from the abnormal blasts after CAR-T-cell therapy. (D) Flow cytometry of peripheral blood obtained on day 35 after CAR-T-cell infusion showing abnormal blasts (orange) without expression of CD19, CD20, CD22, CD24, or cytoplasmic CD79a (not shown). The abnormal blasts were CD33^hi, CD64⁺, and CD4⁺ (not shown), and CD14⁻ and CD34⁻ (not shown), consistent with AML with monocytic differentiation. (E) CGAT identified multiple genomic aberrations in the monoblasts isolated after CAR-T-cell infusion, which were not present in the CD19⁺ lymphoblasts isolated before CAR-T-cell infusion. (F) The percentage of CD19-specific CAR-T cells in CD3⁺ T cells (open circles) and the absolute CAR-T-cell count (squares) in blood on the indicated days after CAR-T-cell infusion are shown for patient 2. (G) All plots show mononuclear cells. Flow cytometry of bone marrow before CAR-T-cell therapy demonstrating CD19⁺ abnormal lymphoblasts (red). At diagnosis, the patient’s abnormal blasts also expressed CD34, CD22 (not shown) without CD4, CD10 (not shown), CD13, significant CD33, CD56, CD64, or CD117. (H) Flow cytometry of bone marrow on day 30 after CAR-T-cell infusion, showing no abnormal CD19⁺ blasts or normal B-cell precursors, but abnormal myeloblasts (orange) that express CD34 with CD4, CD13, bright CD33, CD64 (intermediate), and CD117 (subset), with aberrant expression of CD56 on a major subset.