Measurement of Free Circulating Bcr-Abl Fusion Protein and Its Phosphorylation in Patients with Chronic Myeloid Leukemia.
Authors: Jilani I. , Kantarjian H. , Faraji H. , Gorre M. , Cortes J. , Ottmann O. , Bhalla K. , O’Brien S. , Giles F. , Albitar M. 
Published: October 2019

Abstract
Diagnosis and monitoring of therapy in chronic myeloid leukemia (CML) depend on cytogenetic, FISH, and PCR assays for detecting the fusion bcr-abl gene. However, the there is an inherent variability and difficulty in standardizing quantitative PCR-based assays. We assessed the utility of a simplified immunoassay for measuring levels of Bcr-Abl protein and phosphorylated Bcr-Abl for diagnosis of CML and detection of minimal residual disease (MRD). Bcr-Abl protein was immunoprecipitated on beads with anti-Bcr antibody, and the fusion protein was detected with anti-Abl antibody. Phosphorylation of the immunoprecipitated Bcr-Abl was measured using antibodies against phosphorylated Abl protein at tyr245 and thr735. Because the relatively high turnover of leukemic cells enriches plasma with leukemic DNA, RNA, and protein, as we previously reported, this assay can use cell lysate as well as PB plasma. In vitro studies with denatured K562 cells in plasma showed reliable detection at levels as low as 10 cells/mL plasma. After exposure of cell cultures to 0.5 μM imatinib, K562 cell lysates exhibited a decreased ratio of phosphorylated Bcr-Abl protein:total Bcr-Abl protein with both the tyr245 (from 0.53 to 0.11) and the thr735 (from 0.36 to 0.08) antibodies. The assay was validated by demonstrating negativity (specificity) in 116 normal control subjects (or patients with other translocations) and positivity in all 380 positive samples, as confirmed by FISH or cytogenetics. The CV for inter- and intra-assay variation was <6%. In CML patients receiving imatinib (n=52), the immunoassay yielded Bcr-Abl measurements parallel to those of cell-based RT-PCR. Of 32 plasma and PB cell samples collected at 6, 9, and 12 months of imatinib therapy, 10 were negative by RT-PCR on cells but 4 of these were positive by the plasma immunoassay. Conversely, 9 of the plasma samples were negative by immunoassay, 4 of which were positive by RT-PCR on PB cells. At 3 months of therapy, the immunoassay yielded negative results in 5 of 33 available samples, all of which were positive by RT-PCR on PB cells. Patients with a high level of disease at 3 months of therapy as assessed by RT- PCR (Bcr-Abl:Abl mRNA ratio >0.05) had significantly more relative phosphorylated Bcr-Abl protein than those with low disease activity (P=0.05). In conclusion, these findings demonstrate that Bcr-Abl and phosphorylated Bcr-Abl are detectable at significant levels in the plasma of CML patients and that these levels can be used to monitor MRD. This free circulating Bcr-Abl protein is most likely complexed with other proteins. Unlike PCR-based assays, our ELISA-like assay can be standardized for use in various laboratories. More importantly, the CV of the immunoassay (<6%) is much lower than that typically reported for RT-PCR (up to 30%). We speculate that the use of plasma rather than cells in this assay allows more accurate quantitative measurement of the disease in the whole body, without potential bias due to sampling errors.