Improved Methodology for Detection of Mutations in the BCR-ABL Fusion Gene That Cause Resistance to the Tyrosine Kinase Inhibitor Imatinib.

Abstract Chronic myelogenous leukemia (CML) is caused by a balanced translocation between chromosomes 9 and 22 that creates the BCR-ABL fusion gene (FG) and activates the ABL tyrosine kinase (TK). Imatinib inhibits this TK and causes long-term remission in most CML patients. Relapse during imatinib therapy is often associated with mutations in the BCR-ABL FG that cause resistance to inhibition by imatinib. The usual procedure for detecting imatinib resistance mutations involves reverse transcription, PCR of the BCR-ABL FG, then nested PCR of the ABL TK to generate a template for sequencing. This procedure is suboptimal in a clinical laboratory because of the risk of carryover contamination, and extra time and workload, all due to the use of nested PCR. We describe a procedure for preparing sequencing template from the BCR-ABL FG that does not require nested PCR. The initial PCR employs a mixture of thermostable DNA polymerases that increases the sensitivity of the reaction for limiting concentrations of the desired target. One round of PCR provides enough template for sequencing. The PCR products are sequenced directly without gel purification, using three internal primers plus the downstream PCR primer as sequencing primers. The internal primers provide additional specificity resulting in a high quality sequence even if the PCR produces undesired products. Using this methodology we analyzed a series of eleven CML patient specimens selected because of a four-fold or greater increase in expression of the BCR-ABL FG during treatment with imatinib. The expression of BCR-ABL RNA was measured using a reverse transcription Q-PCR assay. The leftover cDNA from the expression assay was used as template for the PCR to generate the sequencing template. Five patients lacked a detectable mutation, and six had a mutation in the BCR-ABL TK. The six mutations found, G279E, F317L, L248V, M351T, F359C, and H396R, had all been seen previously and associated with resistance to imatinib. We conclude that detection of imatinib resistance mutations in the BCR-ABL FG is feasible without using nested PCR, although with a reduced analytical sensitivity. Elimination of the nested PCR step makes the procedure easier, quicker and less likely to suffer from carryover contamination. This method is a viable alternative for the detection of imatinib resistance mutations in the clinical laboratory.