Abstract

Although tyrosine kinase inhibitor therapy with imatinib in CML patients leads to a high rate of long-term disease control, the development of resistance largely due to bcr-abl point mutations has become a therapeutic challenge. Dasatinib, a recently approved kinase inhibitor, and other new agents invariably have a unique pattern of activity against particular drug-resistant forms of BCR-ABL. It will therefore be helpful to determine the specific mutations that mediate resistance in order to employ the optimal alternative agents in relapsed or refractory patients. As such, there is a need for a rapid, sensitive, and quantitative assay to identify and monitor drug resistance mutations. We have adapted a method for genotyping single nucleotide polymorphisms to mutation detection in clinical samples of CML patients. This “polony” technique entails solid phase PCR amplification of single DNA molecules in a polyacrylamide gel (forming a PCR-colony, or “polony”), followed by discrimination of wild type and mutated nucleotides by a fluorescent single nucleotide base extension assay. The technique is versatile and sensitive (detection limit: 1 mutant in 10−4 wild type cells); it allows us to quantify the BCR-ABL to ABL ratio and to haplotype and screen for several mutations in the same cell population. We have used this assay to screen for the most common BCR-ABL mutations in CML to discern the dynamics of specific mutations following treatment with BCR-ABL kinase inhibitors. One imatinib-treated patient sustained a cytogenetic relapse and was switched to dasatinib. This patient was found to have a T315I mutation that caused subsequent dasatinib resistance. The polony assay detected this mutation from a rare presence to the dramatic takeover in concert with the patient’s rapid disease progression. In another imatinib-treated patient who sustained a cytogenetic relapse on imatinib the polony assay detected an M244V mutation, modestly resistant to imatinib (IC50 3.1μM). Doubling the imatinib dose the patient regained cytogenetic response, which was documented by the polony assay. A third patient who exhibited primary resistance to imatinib was switched to nilotinib after progression to accelerated phase, but without success. The polony assay identified an E255K mutation, known to be quite resistant to imatinib (IC50 5.2μM), relatively resistant to nilotinib (IC50 200nM) but sensitive to dasatinib (IC50 5.6nM). Dasatinib therapy resulted in a rapid hematologic response, with a drop in WBC from 60,000 to 5000, and a complete clearance of blasts. The polony assay showed a rapid decrease of the E255K mutation from a frequency of almost 100 percent to about 20 percent. These patients and others in our clinical surveys illustrate the importance of having a reliable means of detecting and quantifying the mutational burden of patients in order to make informed treatment decisions. A prospective clinical trial testing the role of mutational monitoring in guiding the choice of novel agents is required to prove the likely clinical utility of the polony assay in the current era of burgeoning target-directed therapy in CML.

Disclosure: No relevant conflicts of interest to declare.

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