Myeloid Leukemia

Detection of BCR-ABL Mutations 93
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Detection of BCR-ABL Mutations and Resistance
to Imatinib Mesylate
Susan Branford and Timothy Hughes
Summary
The major mechanism of imatinib resistance for patients with chronic myeloid leukemia
(CML) is clonal expansion of leukemic cells with mutations in the Bcr-Abl fusion tyrosine
kinase that reduce the capacity of imatinib to inhibit kinase activity. The early detection of such
mutations may allow timely treatment intervention to prevent or overcome resistance. Direct
sequencing of the BCR-ABL kinase domain is relatively rapid and allows detection of emerging
mutations at a sensitivity of approx 20%. Mutations have been detected over a range of 242
amino acids, which spans the entire kinase domain. For optimal sensitivity, the kinase domain
of the abnormal gene should be isolated by reverse-transcription (RT) polymerase chain reaction
(PCR) amplification using primers that hybridize to the BCR and ABL genes. The quality
of the RNA is assessed by real-time quantitative PCR prior to analysis, and BCR-ABL levels are
determined. Only RNA of adequate quality is used to ensure accurate and reproducible mutation
analysis. Depending on the level of BCR-ABL transcripts, a one- or two-step PCR is
required to amplify the kinase domain. Direct sequencing with dye terminator chemistry is
performed using PCR-purified products. The sequence is compared to an ABL kinase domain
reference sequence using sequencing analysis software, which aligns the sequences and highlights
single or multiple mutations.
Key Words: Chronic myeloid leukemia; BCR-ABL; imatinib mesylate; mutation; kinase
domain; real-time quantitative PCR; direct sequencing.
1. Introduction
Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome
translocation (t(9;22)). The resultant BCR-ABL fusion gene encodes a
protein with constitutive tyrosine kinase activity that is causally associated with
CML (1–3). The level of BCR-ABL mRNA can be used as a sensitive marker of
disease progress. CML progresses from a relatively benign chronic phase to an
accelerated phase that is characterized by increasing numbers of hematopoietic
cells and additional chromosomal abnormalities. The disease terminates in the
From: Methods in Molecular Medicine, Vol. 125: Myeloid Leukemia: Methods and Protocols
Edited by: H. Iland, M. Hertzberg, and P. Marlton © Humana Press Inc., Totowa, NJ
93