BCR::ABL1 Deep Molecular Response Quantification and Transcript Type Identification in Chronic Myeloid Leukemia Using a US Food and Drug Administration-Approved Droplet-Based Digital PCR Assay

BCR::ABL1 digital PCR is a promising technique for the quantification of deep molecular responses (DMRs) in chronic myeloid leukemia. It provides an improved precision and sensitivity compared with conventional real-time quantitative PCR (qPCR), which is particularly relevant in the context of prediction of successful treatment-free remission. This study assessed the feasibility of BCR::ABL1 digital PCR in clinical practice. A total of 168 DMR samples of patients with chronic myeloid leukemia aiming for a treatment-free remission attempt were assessed by both digital PCR and qPCR. Digital PCR was performed with the droplet-based Bio-Rad QXDx BCR-ABL %IS assay, using eight replicates per sample. qPCR was performed with the fully automized Cepheid Xpert BCR-ABL Ultra assay. Various technical and practical aspects of BCR::ABL1 quantification using digital PCR were assessed. The reported limit of detection of the qPCR is molecular response 4.5, requiring an equivalent of 32,000 ABL1 transcripts. Using digital PCR, a median number of ABL1 of approximately 300,000 were obtained. BCR::ABL1 was quantifiable by digital PCR in 68% of the samples below qPCR's limit of detection. In addition, e13a2 and e14a2 BCR::ABL1 transcript types could be discriminated based on the mean fluorescence intensity of BCR::ABL1-positive droplets. BCR::ABL1 digital PCR is feasible for DMR quantification in clinical practice and offers an increased sensitivity over qPCR. (J Mol Diagn 2025, 27: 109-118; https:// doi.org/10.1016/j.jmoldx.2024.11.003)