Imatinib mesylate (Gleevec, Novartis Pharmaceuticals) is a tyrosine kinase inhibitor (TKI) widely used in the treatment of Chronic Myeloid Leukemia (CML) and Philadelphia Positive Acute Lymphocytic Leukemia (Ph+ALL). Imatinib blocks the ATP binding site in the ABL kinase domain, maintaining the protein in an inactive conformation. Point mutations within the ABL kinase domain prevent the binding of imatinib, resulting in clinical resistance. Identification of ABL kinase mutations can result in early detection of imatinib resistance.
The presence of an ABL kinase point mutation is closely linked to the development of TKI (imatinib, dasatinib, or nilotinib) resistance. These mutations are found in roughly one half of cases of TKI resistance. In some cases, however, ABL mutations can be detected at low levels in a patient responding to TKI therapy, and do not necessarily evolve into resistance. Thus, the detection of an ABL mutation must be placed in clinical context. More than 80 point mutations spanning the entire ABL kinase domain have been detected, and these mutations yield varying degrees of resistance to the TKIs.
T315I and P-loop mutations are strongly linked to resistance, blast crisis and relapse. Specifically, T315I is associated with poor outcome and confers complete resistance to imatinib and other generations of TKI therapies.
Peripheral blood or bone marrow aspirates are extracted to RNA and the BCR/ABL transcript is amplified by reverse transcription polymerase chain reaction (RT-PCR). The ABL kinase domain is further amplified in a nested PCR reaction and is sequenced using Dye terminator chemistry. The ABL sequence is then compared to a wild-type reference sequence using Mutation Surveyor (SoftGenetics). Detection of ABL mutations occur at a sensitivity of 25-30% and all mutations are confirmed with a second primer.