Abstract Lineage switch is a rare phenomenon, occurring in less than 1% of leukemia cases. However, with the advent of CD19-targeted immunotherapies such as CAR-T and blinatumomab, it has emerged as a significant complication. Identifying patients at high risk for lineage switch prior to immunotherapy is critical, yet predictive biomarkers remain unclear. In a cohort of 65 B-ALL patients who relapsed after CAR-T therapy, we identified one case of myeloid lineage switch with unknown mechanism. Transcriptomic sequencing across three timepoints: pre-CAR-T, first CAR-T relapse, and second CAR-T relapse (myeloid switch) confirmed a consistent driver gene profile (ETV6::RUNX1 fusion, NF1, WNK1 and CIC mutations), ruling out a secondary malignancy. To elucidate the mechanism, we performed single-cell DNA-protein sequencing (Mission Bio Tapestri platform) and single-cell RNA sequencing (10x Genomics platform). At first CAR-T relapse, we identified five distinct clones. After CAR-T therapy, three clones were undetectable, whereas two clones persisted and underwent rapid expansion, concomitant with myeloid lineage switch. Notably, both of these clones harboring mutations in TP53 and epigenetic regulator STAG2. We further discovered a distinct population of lineage-infidelity cells (CD117+CD71+ CD34+CD38+CD56+CD19dim) that co-express myeloid and B-lineage markers, representing a heightened plasticity that may facilitate CAR-T evasion. Projection of our scRNA-seq data onto a normal developmental atlas showed pre-CAR-T sample from the lineage-switch case encompassed a broad spectrum, whereas control case of CD19-negative relapse due to CD19 mutation were confined to the pro-B/pre-B stages. Based on these findings, we hypothesized that co-mutation of TP53 and epigenetic regulators predisposes cells to lineage switch. To validate this finding, we performed a longitudinal flow cytometry analysis. Patients were stratified into two groups: a co-mutation group (n=4) harboring TP53 and epigenetic gene mutations, and a control group (n=2) lacking these mutations. The co-mutation group exhibited significant downregulation of the B-lineage marker CD19 and concurrent upregulation of the myeloid marker following CAR-T treatment. In contrast, the control group showed no notable changes. In conclusion, our data suggests that co-mutations in TP53 and epigenetic genes enhance plasticity in B-ALL. Under the selective pressure of CD19 CAR-T therapy, subclones harboring these mutations are preferentially expanded, leading to lineage switch. We propose monitoring myeloid marker expressing subclones in B-ALL patients carrying co-mutations in TP53 and epigenetic genes during immunotherapy is important to improve their prognosis. Citation Format: Shuang Zhao, Yanjing Tang, Meng Su, Bowen Cui, Rongrong Fan, Han Wang, Liu Yang, Lixia Ding, Ronghua Wang, Huiying Sun, Ying Zhong, Qiaoqiao Shi, Yuxuan Guo, Lili Song, Xinyu Wan, Tianyi Wang, Jing Yang, Benshang Li, Yu Liu. Single-cell multi-omics reveals co-mutation of TP53 and epigenetic gene driving myeloid transformation in B-ALL following CAR-T therapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5248.
Zhao et al. (Fri,) studied this question.