Double-Gradient–Denaturing-Gradient Gel Electrophoresis for Mutation Screening of the BCR-ABL Tyrosine Kinase Domain in Chronic Myeloid Leukemia Patients

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Chronic myeloid leukemia (CML) is a rare hematopoietic stem cell disorder characterized by the t(9;22) translocation. This somatic event leads to the formation of the BCR-ABL fusion gene, which is translated in a functional protein (1). The Bcr-Abl oncoprotein differs from the endogenous c-Abl protein in both its subcellular localization and its tyrosine kinase (TK) activity. The deregulated Abl TK activity of the Bcr-Abl protein initiates the oncogenic process and represents a target to TK inhibitors. Among a series of compounds exhibiting TK inhibition, STI 571 (imatinib) was found to be highly effective against Abl and its derivative Bcr-Abl (2)(3). This molecule targets the inactive conformation of the kinase, which leads to stabilization of the protein in its inactive form and impairs ATP binding (4). Approved for the treatment of CML, imatinib induces hematologic and cytogenetic remissions in the chronic phase as well as in the blast phase (5)(6)(7). A significant proportion of patients, however, become resistant to the treatment. Different mechanisms of imatinib resistance have been described, in particular point mutations within the sequence of the BCR-ABL gene coding for the TK domain (8)(9)(10). Approximately 30 missense mutations have been identified in CML patients. Within the Bcr-Abl TK domain, these mutations are located in the nucleotide binding loop (P-loop), in the active site (imatinib contact site), or in the activation loop (A-loop; Fig. 1A⇓ ). Among these mutations, some prevent the binding of imatinib, and others make this binding more difficult. In the latter case, a dose increase can overcome the resistance. Recently, a novel TK inhibitor was described (11). In vitro, this molecule remains active for the majority of Bcr-Abl mutants, with the exception of T315I. Molecular monitoring of CML patients is based on the quantification …