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 BioRad QXDx BCR-ABL % International Scale 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, we were able to obtain a median number of ABL1 of approximately 300,000. BCR::ABL1 was quantifiable by digital PCR in 68% of the samples below qPCR's limit of detection. In addition, we observed that 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.