Abstract
Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.
Highlights
Chronic myeloid leukemia (CML) originates from a pluripotent hematopoietic stem cell (HSC) that acquires the t(9;22)(q34;q11) translocation, i.e. the hallmark cytogenetic aberration of CML
Conditions for detecting BCR-ABL1 fusion proteins through in situ PLA with flow cytometry as readout, PLA-flow (Fig. 1), were established using K562 cells that carry the BCR-ABL1 fusion, while the BCR-ABL1 negative cell line U937 was used as a negative control
Cells positive for the BCR-ABL1 fusion protein were detected in samples from CML patients, while healthy control samples lacked labeled cells (Fig. 2)
Summary
Chronic myeloid leukemia (CML) originates from a pluripotent hematopoietic stem cell (HSC) that acquires the t(9;22)(q34;q11) translocation, i.e. the hallmark cytogenetic aberration of CML This translocation, commonly referred to as the Philadelphia (Ph1) chromosome, leads to a fusion protein, where the 5′ part of the BCR gene is fused to the 3′ part of the ABL1 gene[1,2,3,4], results in significantly increased tyrosine kinase activity and cell proliferation. We describe a novel approach to monitor CML patients by quantifying leukocytes harboring the BCR-ABL1 fusion at the protein level This method, called PLA-flow, uses the in situ proximity ligation assay (in situ PLA)[18, 19] to detect the BCR-ABL1 fusion protein inside cells (Fig. 1). Usage of flow cytometry gives the advantage of simultaneously investigation of surface markers commonly applied in the clinical routine setting
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