Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

Abstract

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

Evaluation of a new flow cytometry based method for detection of BCR-ABL1 fusion protein in chronic myeloid leukemia

“…In the present study 4 MRD patients escaped the detection of BCR-ABL fusion protein by present flow cytometric method. Otherwise, in case of first diagnosis and monitoring the follow up cases, flow cytometry can be run reliably and safely which is easier, quicker and simpler, and fully translatable to routine management of CML patients [ 26 ].…”

Technical challenges for standardization of molecular monitoring in chronic myeloid leukaemia in developing countries

“…Molecular testing for the BCR-ABL fusion gene provides important prognostic information for individual chronic myeloid leukaemia (CML) patients treated with tyrosine kinase inhibitors. International treatment recommendations include specific time-dependent molecular milestones to assess whether optimal molecular response is achieved (Huet et al, 2014;Recchia et al, 2015). To date, real-time quantitative polymerase chain reaction (RQ-PCR) is considered the gold standard method to measure the level of BCR-ABL mRNA relative to an internal reference gene (Cross et al, 2015a).…”

A Prognostic and Predictive Study of BCR-ABL Expression Based on Characterization of Fusion Transcripts

“…Chronic myeloid leukemia is a disorder of hematopoietic stem cell that results in increased myeloid cells within the peripheral blood, directing it to myeloid hyperplasia in bone-marrow which affects the erythroid cell as well as the platelet count [1]. This infirmity is a result of translocation that juxtaposes the ABL gene (Abelson) on the BCR gene (breakpoint cluster region) forming a BCR-ABL mutation also known as Philadelphia Chromosome, which primes to increased proliferation of leukemic cells [2]. The chimeric protein p210 thereby formed as a result of this translocation, constitutively leads to activation of tyrosine kinase activity [3].…”