BCR-ABL Protein Detection by Flow Cytometry in Acute Lymphoblastic Leukemia Samples

The development of agents directed specifically against the BCR-ABL tyrosine kinase has opened therapeutic avenues that have profoundly modified the management of diseases which harbor the genetic abnormality that leads to the production of the BCRABL transcript. While the role of tyrosine kinase inhibitors of 1^st and 2^nd generation in chronic myeloid leukemia (CML) patients is well established, important clinical responses have been documented also in BCR-ABL+ acute lymphoblastic leukemia (ALL), the most relevant genetic ALL subgroup which accounts for about 25% of adult cases and is associated with a dismal prognosis. Following the administration of tyrosine kinase inhibitors alone as induction treatment, complete hematologic remissions have in fact been recorded in virtually all cases. Thus, the possibility of identifying the presence of the BCR-ABL transcript in the shortest possible time and with a simple and broadly utilizable technique would translate into the timely implementation of a targeted therapy. The availability of a method capable of highlighting the presence of the BCR-ABL protein has important implications because it can document the effective transduction of the molecular transcript; since tyrosine kinase inhibitors are effective on the protein, this would unequivocally support the use of such specific agents in these cases. For this purpose, we have utilized the BCR-ABL Protein Kit^* (BD Biosciences, San José, CA) which consists of an immunoassay that identifies qualitatively the presence of the BCR-ABL protein in the lysates of leukemic cell populations. The BCR-ABL fusion protein is captured and detected on Cytometric Bead Array (CBA) and analyzed on a FACSCanto (BD Biosciences) flow cytometer. Lysates from normal mononuclear cells and from the K562 cell line were used as negative and positive controls, respectively. The presence of the BCR-ABL protein was investigated on fresh bone marrow or peripheral blood samples from 56 consecutive cases of acute leukemia aged between 11 and 81 years referred to our Institution. The BCR-ABL protein could be documented in 11 of the 56 cases analyzed. By conventional immunophenotypic analysis they proved to be a B-lineage ALL in 9 cases and a B lymphoid blast crisis of CML in 2. In all 11 cases, the presence of the BCR-ABL transcript was subsequently documented by molecular biology. In the other 45 cases the diagnosis was: B-lineage ALL in 26 (2 of which were ALL/AF4+), T-ALL in 2, acute myeloid leukemia in 22 (5 of which were PML-RARα+) and acute biphenotypic leukemia in 1. None of these latter 45 cases harbored the BCRABL transcript. The assay utilized was successfully completed within a maximum of 4 hours from the marrow or blood collection on 20 × 10⁶ cells containing at least 20% of leukemic cells. The correlation between the presence/absence of the BCR-ABL protein evaluated using the BCR-ABL Protein Kit^* and the positivity/negativity of the transcript assessed according to conventional genetic techniques has been absolute. In our laboratory, the assay has shown to be reliable, reproducible and of simple execution. It allows in a very short time to identify the presence of the BCR-ABL protein – both p190 and p210 – through a flow cytometric analysis and thus to reliably offer proteomic information to investigators researching targeted anti-tyrosine kinase treatment.