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CHRONIC MYELOGENOUS LEUKEMIA

Droplet digital PCR for the detection of second-generation tyrosine kinase inhibitor-resistant BCR::ABL1 kinase domain mutations in chronic myeloid leukemia

Abstract

One of the indications for BCR::ABL1 mutation testing in chronic myeloid leukemia (CML) is when tyrosine kinase inhibitor therapy (TKI) needs to be changed for unsatisfactory response. In this study, we evaluated a droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harbouring mutations conferring resistance to second-generation TKIs (2GTKIs). Parallel quantitation of e13a2, e14a2 and e1a2 BCR::ABL1 fusion transcripts enables to express results as percentage of mutation positive- over total BCR::ABL1 transcripts. We determined the limit of blank in 60 mutation-negative samples. Accuracy was demonstrated by further analysis of 48 samples already studied by next generation sequencing (NGS). Mutations could be called down to 0.5% and across 3-logs of BCR::ABL1 levels. Retrospective review of BCR::ABL1 NGS results in 513 consecutive CML patients with non-optimal response to first- or second-line TKI therapy suggested that a ddPCR-based approach targeted against 2GTKI-resistant mutations would score samples as mutation-negative in 22% of patients with warning response to imatinib but only in 6% of patients with warning response to 2GTKIs. We conclude ddPCR represents an attractive method for easy, accurate and rapid screening for 2GTKI-resistant mutations impacting on TKI selection, although ddPCR cannot identify compound mutations.

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Fig. 1: Schematic representation of the expected output for tube 1, 2, and 3.
Fig. 2: Representative results obtained with serial dilutions of mutated into unmutated BCR::ABL1-positive cell lines.
Fig. 3: Results obtained with serial dilutions of mutated into unmutated BCR::ABL1-positive cell lines and BCR::ABL1-negative cell lines mimicking different BCR::ABL1/ABL1 transcript levels.
Fig. 4: Representative examples of 2D plots.
Fig. 5: Mutation kinetics by ddPCR and by NGS in three representative patients who developed 2GTKI-resistant mutations on therapy.

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Materials described in this manuscript, including all relevant raw data, will be freely available upon request to any researcher wishing to use them for non-commercial purposes.

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Acknowledgements

The study was supported by Novartis through the European Treatment and Outcome Study (EUTOS) for CML. ddPCR assays were provided by Bio-Rad Laboratories.

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SS: designed the study, analyzed and interpreted results, contributed to writing the report; SDS, MMartelli, CM, SB, MMancini, CV: performed the study, contributed to data analysis and interpretation, prepared the figures and tables; FC, GG, CP: performed patient and sample selection, contributed to data analysis and interpretation; KMP, TE: provided important study materials, contributed to data analysis and interpretation, contributed to writing the report; DM, AC: provided reagents, contributed to experimental protocol set up and optimization; MC, designed the study, coordinated the clinical and research groups, contributed to writing the report. All authors read and approved the manuscript for submission.

Corresponding author

Correspondence to Simona Soverini.

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Competing interests

ddPCR assays for this study were provided by Bio-Rad Laboratories. Bio-Rad Laboratories had no role in the design of the study, collection and analysis of data and decision to publish. DM and AC are Bio-Rad’s employees. The remaining authors declare no conflicts of interests.

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Soverini, S., De Santis, S., Martelli, M. et al. Droplet digital PCR for the detection of second-generation tyrosine kinase inhibitor-resistant BCR::ABL1 kinase domain mutations in chronic myeloid leukemia. Leukemia (2022). https://doi.org/10.1038/s41375-022-01660-8

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