Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure. We used single-cell RNA sequencing (scRNAseq) approach to investigate leukemic cells of one B-ALL patient at two-time points; before (T1) and after (T2) anti-CD19 CAR-T cell therapy. At T2 leukemic cells were CD19 negative (CD19neg). Therefore, we asked whether these CD19neg B-ALL clones were present before CAR-T cell therapy. To address this issue, T1 and T2 samples were sorted according to forward scatter and side scatter, a cell viability marker, CD3 and CD19 surface expression. We obtained 4 tubes of sorted cells: T1-CD19pos, T1-CD19neg, T2-CD19pos and T2-CD19neg. Each of those samples were marked with a distinct anti-CD45-hashtag oligonucleotide (HTO) antibody, then mixed and analyzed by scRNAseq using the 5’ 10X Genomics technology.