Detection of Drug-Resistant Clones in Chronic Myelogenous Leukemia Patients during Dasatinib and Nilotinib Treatment

Abstract

Imatinib effectively inhibits the tyrosine kinase activity conferred by the BCR-ABL gene [fusion gene of BCR (breakpoint cluster region) and ABL1 (c-abl oncogene 1, receptor tyrosine kinase)] and thereby appreciably improves outcomes for chronic myelogenous leukemia (CML). A small percentage of patients relapse because of the proliferation of escape clones; such relapses can be treated with second-generation drugs. Early detection and monitoring of resistant clones may provide clinical benefit. We describe the development and testing of a new approach for quantitative monitoring of CML resistance. We designed mutation-specific assays that use hydrolysis probes and an array of allele-specific primers containing nucleotides mismatched at various positions. All assays were tested with plasmids containing corresponding mutant or wild-type sequences, allowing identification of optimal assays for specific and effective amplification of the target template. Clinical samples were then used to compare the results of selected assays with those of standard genotyping. We used a modified amplification refractory mutational system approach and testing with plasmid constructs to design assays that allowed highly selective detection of resistance for all target mutations. By taking advantage of single-step performance and high PCR efficiency, we were able to quantitatively track the absolute amount of resistance conferred by a specific mutation over 4 orders of magnitude. Moreover, we designed an integrated test for dasatinib resistance that uses multiple primers simultaneously. These single-step, closed-tube assays specifically target mutations associated with resistance to dasatinib or nilotinib. Compared with standard genotyping, such biased genotyping improves the detection of resistance or alternative features via quantitative analysis of the absolute amount of resistance.