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.

Abteilung Hämatologie Onkologie Klinik für Innere Medizin 2 Universitätsklinikum Jena Jena Germany

Bio Rad Laboratories Digital Biology Group Peterborough UK

Bio Rad Laboratories Digital Biology Group Pleasanton CA USA

Dipartimento di Medicina Specialistica Diagnostica e Sperimentale Istituto di Ematologia Seràgnoli Università di Bologna Bologna Italy

Institute of Hematology and Blood Transfusion Prague Czechia

IRCCS Azienda Ospedaliero Universitaria di Bologna Istituto di Ematologia Seràgnoli Bologna Italy

IRCCS Azienda Ospedaliero Universitaria di Bologna Istituto di Ematologia Seràgnoli Dipartimento di Medicina Specialistica Diagnostica e Sperimentale Università di Bologna Bologna Italy

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2025 European LeukemiaNet recommendations for the management of chronic myeloid leukemia

European LeukemiaNet laboratory recommendations for the diagnosis and management of chronic myeloid leukemia