Flow Cytometric Detection of BCR-ABL Fusion Proteins in Leukemia Patients Via An Immunobead Assay
Authors: Weerkamp Floor , Dekking E. , Van der Velden Vincent H.J. , Böttcher Sebastian , Boeckx Nancy , Van Wering Elisabeth R. , Montero Juan Flores , Lucio Paulo , Sedek Lukasz , Kalina Tomas , Macintyre Elizabeth , Staal Frank J.T. , van Dongen Jacques JM 
Published: April 2020

Abstract
The BCR-ABL fusion gene results from the translocation t(9;22). It is the hallmark of chronic myeloid leukemia (CML) and is present in a poor-risk subgroup of precursor B cell acute lymphoblastic leukemia (ALL), which represents 25–30% of adult ALL and 3–5% of childhood ALL. Consequently the detection of the BCR-ABL aberration is of utmost importance for diagnosis and classification of leukemia patients and can also be used as marker for monitoring of BCR-ABL+ leukemias to evaluate treatment effectiveness. So far, the BCR-ABL aberration has been detected by cytogenetics, FISH or PCR, all techniques that are time consuming and require special facilities. We developed a simple flow cytometric bead assay for detection of the BCR-ABL fusion protein in cell lysates, using a bead-bound catching antibody against one side of the fusion protein and a fluorochrome-conjugated detection antibody against the other side of the fusion protein. The anti-BCR antibody was developed against a non-homologous region of ~80 amino acids, encoded by exon 1 in order to detect all know BCR-ABL variants, including p190, p210, and p230. The assay appeared to be specific and sufficiently sensitive to detect BCR-ABL proteins in leukemic cell lysates. However, protein stability problems were encountered when cell samples contained high frequencies of mature myeloid cells with high levels of protease activity, such as CML cells and granulocyte fractions. This problem could be significantly reduced by adding protease inhibitors to several steps of the immunobead assay.
The immunobead assay was further developed and standardized by BD Biosciences into the Cytometric Bead Array (CBA) assay (BCR-ABL Protein Kit; BD Biosciences, San Jose, CA) for Research Use Only. Large scale testing of this kit in 9 diagnostic laboratories of the EuroFlow Consortium showed that the results of the flow cytometric BCR-ABL immunobead assay were fully concordant with the PCR results in a series of 83 freshly collected samples from newly diagnosed (n=75) or relapsed (n=8) leukemia patients: 13 CML patients, 53 ALL patients, and 17 acute myeloid leukemia (AML) patients (see table). All blood samples tested from 61 healthy controls were negative in the immunobead assay.
BCR-ABL PCR assay BCR-ABL immunobead assay negative p190 p210 negative* weak* medium* high* * negative: MFI value &lt;150; weak: MFI value ≥150 and &lt;500; medium: MFI value ≥500 and &lt;2000; high: MFI value ≥2000 Healthy controls (n=61) NT 61 0 0 0 Precursor-B-ALL - childhood (n=22) 22 0 0 22 0 0 0 - adult (n=19) 7 9 3 7 1 1 10 T-ALL (n=12) 12 0 0 12 0 0 0 AML (n=17) 17 0 0 17 0 0 0 CML (n=13) 1 1 11 1 5 4 3
We conclude that the flow cytometric immunobead assay is a fast and easy technique for specific detection of BCR-ABL proteins in leukemic cells. The assay is independent of the breakpoint position in the BCR gene, does not need special laboratory facilities other than a routine flow cytometer, provides results within several hours, and can be run in parallel to routine immunophenotyping. Since differentially labeled beads allow multiplexing, it will be possible to develop single tube assays for combined evaluation of multiple different fusion proteins, e.g. per disease category. Consequently, the flow cytometric immunobead assay can contribute to fast and easy diagnosis and classification of leukemias. If sufficient sensitivity can be reached, also monitoring of minimal residual disease becomes possible.