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Molecular Diagnostics

Innovative rapid screening for complex BCR-ABL1 kinase ___domain mutations in TKI-treated leukaemia patients

British Journal of Cancer (2025)Cite this article

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Abstract

Background

Screening for BCR-ABL1 kinase ___domain (KD) mutations is routinely implemented in cases of treatment failure for chronic myeloid leukaemia and Philadelphia-positive acute lymphoblastic leukaemia. However, timely deciphering their clonal relationship via mutation profiling that requires identification of mutation types, quantification of mutant abundance, and differentiation between compound and polyclonal mutations (CMs and PMs), remains difficult during therapy.

Methods

Herein, we established a protocol that identified mutation types and further distinguished clonal relationships by combining mini-sequencing of MeltArray with allele segregation of droplet digital PCR (ddPCR).

Results

The analysis showed that 78 samples (18.93%) were mutant, of which 50 (64.1%) harboured single mutations, and 28 (35.9%) contained multiple mutations, including double-, triple-, quadruple- and hepta-mutants. These results agreed with NGS, except one sample with F317L and L324Q mutations, where L324Q was beyond MeltArray’s scope. Among cases containing multiple mutations, 85.71% were PMs, 10.71% were CMs, and 3.57% were mixed CMs and PMs. Retrospective analysis revealed that clonal relationships in BCR-ABL1 KD mutations were highly dynamic during therapy.

Conclusions

The MeltArray-ddPCR protocol enables dynamic profiling of BCR-ABL1 KD mutations to determine clonal status, improving prediction of drug susceptibility and leukaemia outcomes.

In this study, we performed a retrospective analysis of 539 samples from 365 leukaemia patients. We developed a comprehensive BCR-ABL1 kinase ___domain mutation screening protocol that includes pre-amplification, mutation screening, and differentiation between compound mutations (CMs) and polyclonal mutations (PMs). By integrating MeltArray and ddPCR technologies, this protocol enables dynamic monitoring of changes in mutation levels and clonal evolution. It also offers companion diagnostics based on mutational profiles to guide precision therapy for leukaemia patients.

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Fig. 1: Schematic diagram of MeltArray-ddPCR protocol.
Fig. 2: Mutational signatures in the mutant samples.
Fig. 3: Three schemes of drop-off probe-based ddPCR assay and looping-out probe-based melting curve analysis.
Fig. 4: Mutational signatures in multi-mutant samples.
Fig. 5: MeltArray-ddPCR protocol for the detection of patient 1 at different timepoints.
Fig. 6: Dynamic change of BCR-ABL1/ABL1 on the IS alongside different clonal relationships before and after transplantation.

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Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

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Acknowledgements

We thank the patients with leukaemia and their families for providing their specimens for the advancement of cancer research.

Funding

This study was supported by the National Natural Science Foundation of China (82271729); the Natural Science Foundation of Xiamen, China (3502Z20227172); the Science and Technology Planning Projects of Liuzhou City (2023YRZ0103); and the Natural Science and Technology Major Project of Xiamen (3502Z20231034).

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Author notes

  1. These authors contributed equally: Yanping Lan, Jun Deng.

Authors and Affiliations

  1. Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China

    Yanping Lan, Pujing He, Wen Huang, Anqi Zhu, Xiaoxia Guo, Qiuying Huang & Qingge Li

  2. Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

    Jun Deng, Lv Xiong & Yu Hu

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  1. Yanping Lan
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Contributions

QL, QH, and YL conceived and design the project. YL, PH, WH, AZ, and XG performed the experiments. JD and LX collected peripheral blood and/or bone marrow samples from leukaemia patients. YL and JD collected, analysed and interpreted the data. YL, QH and QL drafted the manuscript. QL, QH, JD, and YH accessed and verified the data. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Qiuying Huang, Yu Hu or Qingge Li.

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The authors declare no competing interests.

Ethics

This study was approved by the Medical Ethics Committee of Union Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology (UHCT-IEC-SOP-016-03-01) and performed in accordance with the Declaration of Helsinki.

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Lan, Y., Deng, J., He, P. et al. Innovative rapid screening for complex BCR-ABL1 kinase ___domain mutations in TKI-treated leukaemia patients. Br J Cancer (2025). https://doi.org/10.1038/s41416-025-03098-y

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