Resistance to molecularly targeted chemotherapy, and the development of novel agents that are active against resistant forms of target proteins create the need for a sensitive and quantitative assay to monitor drug-resistant mutations in patients to guide treatment and assess response. Here, we describe an application of the polymerase colony (polony) method to identify and quantify known point mutations in the BCR-ABL oncogene in patients with chronic myelogenous leukemia who evolve resistance to ABL kinase inhibitors. The assay can detect mutations with a sensitivity of 10−4, quantify the burden of drug-resistant cells, and simultaneously monitor the dynamics of several coexisting mutations. As a proof of concept, we analysed blood samples from three patients undergoing therapy with ABL kinase inhibitors and found that the patients' response to therapy correlated with our molecular monitoring. We were also able to detect mutations emerging in patients long before clinical relapse. Therefore, the polony assay could be applied to a larger patient sample to assess the utility of early mutation detection in patient-specific treatment decisions. Finally, this methodology could be a valuable research tool to shed light on the natural behavior of mutations pre-existing kinase inhibitors therapy and either disappearing over time or slowly taking over.
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We are very grateful to Dr DM Sabatini for his critical comments and advice, to D Wine for assistance in image analysis, and to Dr D DeAngelo, Dr MWadleigh and RNP Ilene Galinsky for assistance with coordination of patient samples. We acknowledge Novartis and Bristol Myers Squibb for their support in clinical trials with nilotinib and dasatinib, respectively. This work was supported by grants from the NIH (T32 HL066987-06, VN; F32-CA101505, TR; R01 CA86691, GQD), the NIH Directors Pioneer Award, the Burroughs Wellcome Fund (GQD) and startup funds from the Duke Institute for Genome Sciences and Policy and Basil O'Connor award from March of Dimes (JZ). The authors declare no competing financial interests.
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Nardi, V., Raz, T., Cao, X. et al. Quantitative monitoring by polymerase colony assay of known mutations resistant to ABL kinase inhibitors. Oncogene 27, 775–782 (2008). https://doi.org/10.1038/sj.onc.1210698
- mutations detection
- solid-phase PCR
- drug resistance
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