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IMMUNOTHERAPY

The dynamic evolution of lineage switch under CD19 CAR-T treatment in non-KMT2A rearranged B-ALL patients

Leukemia volume 39, pages 238–242 (2025)Cite this article

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Reconstructing the clonal evolution paradigm helps us understand the process of lineage switching [6]. Therefore, we depicted the clonal evolution pattern of patient 1 (P01) through single-cell targeted DNA sequencing (Supplementary Table 3). Through single-cell genomic sequencing and quality control, we obtained a total of 6566 high-quality cells with related gene mutations (FLT3, BCORL1, and STAG2) at the four time points for clone structure inference (Fig. 1B, Supplementary Table 4). The Tapestri insights analysis (Fig. 1B) revealed that the FLT3-ITD mutation consistently persisted at four different time points (83.3%, 69.4%, 98.8%, 9.0%). Pre-existing BCORL1 mutation rapidly expanded after the initiation of myeloid relapse, while STAG2 mutation occurred with the presence of BCORL1 mutation (16.1%, 4.3%, 96.1%, 0.0%). Compared with the T1_Pre_CART time point, we found that the BCORL1 mutation burden (Fig. 1C) increased at the T3_Relapse time point. Moreover, the single-cell membrane protein data (Supplementary Fig. 1A–D, Supplementary Table 5) showed that B-ALL blast cells expressed typical B-ALL-associated markers CD19 and CD10, along with co-expression of myeloid-associated markers CD33 and CD123 at the T1_Pre_CART time point and the blast cells lost the expression of lymphoid marker CD19 at relapse, confirming the phenomenon of lineage switch.

Moreover, the clonal evolution structure of patient 2 (P02) was reconstructed through whole exon sequencing and targeted sequencing (Fig. 1D, Supplementary Table 6). Throughout the treatment course of P02, the expression of fusion gene EP300::ZNF384 persisted, the IKZF2 mutated clone disappeared after CD19 CAR-T therapy, and the BCOR gene mutated clone emerged upon myeloid lineage relapse. Through BCR sequencing, we observed the presence of identical immunoglobulin sequences at the T2_Relapse time point as those in the T1_Pre_CART time point (Fig. 1E, Supplementary Table 7), and we could also observe that the clonal frequency of immunoglobulin sequences increased during relapse, suggesting their enrichment in the myeloid reprogramming process. This result suggested that the origin of myeloid progenitor cells was reprogrammed from B-ALL cells [7].

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Fig. 1: The genomic alterations of two non-KMT2A-r lineage switch patients.
Fig. 2: The single-cell transcriptomics profiling of P01 and P02.

Data availability

Our raw sequence data (including WES, scRNA-seq, scDNA-seq, membrane protein sequencing, and BCR-seq) have been deposited in the Genome Sequence Archive for Human in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number HRA005240 (https://ngdc.cncb.ac.cn/gsa-human/). The human healthy donors' bone marrow data are obtained from GSE120221 [12].

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Acknowledgements

This work was supported by National Key Research and Development Program of China (2021YFC2500300), CAMS Innovation Fund for Medical Sciences (2021-I2M-1-041), National Natural Science Foundation of China (82000131, 82341213), Clinical Research Foundation of National Clinical Research Center for Blood Diseases (2023NCRCA0101), Tianjin Municipal Science and Technology Commission Grant (23JCYBJC01050, 23JCYBJC01060).

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  1. These authors contributed equally: Shaowei Qiu, Yihan Mei, Runxia Gu.

Authors and Affiliations

  1. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300020, China

    Shaowei Qiu, Yihan Mei, Runxia Gu, Yu Liu, Manling Chen, Haiyan Xing, Kejing Tang, Zheng Tian, Qing Rao, Donglin Yang, Aiming Pang, Shuning Wei, Yujiao Jia, Huijun Wang, Sizhou Feng, Hui Wei, Ping Zhu, Min Wang, Ying Wang, Wenbing Liu & Jianxiang Wang

  2. Tianjin Institutes of Health Science, Tianjin, 301600, China

    Shaowei Qiu, Yihan Mei, Runxia Gu, Yu Liu, Manling Chen, Haiyan Xing, Kejing Tang, Zheng Tian, Qing Rao, Donglin Yang, Aiming Pang, Shuning Wei, Yujiao Jia, Huijun Wang, Sizhou Feng, Hui Wei, Ping Zhu, Min Wang, Ying Wang, Wenbing Liu & Jianxiang Wang

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  1. Shaowei Qiu
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Contributions

SQ, RG, YW, WL, and JW conceived the study design. YM, YL, MC, HX, KT, ZT, and QR conducted the investigation. SQ, YM, WL, and PZ performed the analysis. DY, AP, SW, YJ, HW, SF, HW, and YW provided the resources. YM and WL managed data curation. YM and WL wrote the original draft. SQ, RG, MW, and JW reviewed and edited the manuscript. SQ, RG, and JW acquired funding. SQ and JW supervised the study.

Corresponding authors

Correspondence to Ying Wang, Wenbing Liu or Jianxiang Wang.

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JW: Advisor for AbbVie. Other authors declare no competing interests.

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Qiu, S., Mei, Y., Gu, R. et al. The dynamic evolution of lineage switch under CD19 CAR-T treatment in non-KMT2A rearranged B-ALL patients. Leukemia 39, 238–242 (2025). https://doi.org/10.1038/s41375-024-02449-7

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