Quantitative Monitoring of Mutant BCR-ABL Transcript Levels through Pyrosequencing Analysis.

Resistance to imatinib has become increasingly prominent, especially in later stages of CML. Mutations in ABL kinase domain of the BCR-ABL gene were found in 40% to 90% of the Gleevec-resistance patients. Those mutations can causes changes in various sites on the ABL kinase and, consequently, confer significantly variable levels of resistance to Imatinib, Nilotinib and Dasatinib. The majority of studies on BCR-ABL mutations analyzed the ABL kinase domain of BCR/ABL allele using DNA sequencing. DNA sequencing has its limitation in sensitivity due to background noise and only providing a “yes/no” signal for the detection of mutant BCR/ABL. We established a pyrosequencing assay for the quantitative detection of 16 known mutations, which account for greater than 90~95% of the reported imatinib mutations. The mutations are: T315I, F317L, H396R, G250E, L248V, M244V, Q252H (2 separate point mutations), Y253H, E255V, M351T, F359V, F359V, F359C and E355G. The assay’s sensitivity could detect down to 5~10%. The data showed that our pyrosequencing assay could quantify the relative amount of each allele very accurately on twelve of the BCR-ABL mutant RNA controls, which are highly linear from 10 to 100% BCR-ABL mutant mixture. The intra-run CV was less than 2% in all of the pyrosequencing reactions when a positive sample was repeated 8 times in the pyrosequencing assay. The inter-run CV was less than 5% when three separate pyrosequencing were performed on four RNA samples. 28 samples from CML patients with t (9;22) translocation were analyzed for the presence of the mutations using both the pyrosequencing assay and the traditional DNA sequencing. 10 out of 28 patients had mutations detected by the pyrosequencing assay. The mutations in the ABL kinase were confirmed by DNA sequencing in all of the 10 samples. To further validate the assay’s specificity, twelve mutant BCR/ABL RNAs made by in vitro transcription were subject to pyrosequencing assay and DNA sequencing. The pyrosequencing results agreed 100% with the DNA sequencing results. Our pyrosequencing assay was a sensitive, robust, and accurate quantitative test for the detection of the mutant BCR/ABL and the monitoring of the mutant BCR/ABL transcript levels. The pyrosequencing system is not widely used in clinical and research labs because it is relative expensive, relatively few people are familiar with the system, and its limitation in quantifying adjacent mutation alleles or mutations located in the homopolymer sequences region. We successfully designed pyrosequencing assay for the detection and the quantification of the mutations in the P-Loop and M351 clusters, which have multiple mutations in the cluster, adjacent mutation alleles and have G/C rich sequences. To the best of our knowledge, only one publication reported the quantitative detection of the mutations of ABL kinase of BcrAbl through pyrosequencing, However, we encountered difficulty in repeating the detection of the P-Loop and M351 cluster in our lab using the procedure from the publication. In conclusion, our pyrosequencing assay for Gleevec-resistant mutations provides a reliable quantitative method for the detection of the mutant BcrAbl transcripts. It provides significant value for the early detection of the mutations and for the follow up of CML patients on imatinib. It is an important step for the monitoring of CML patients with Gleevec-resistance because it transitions the measuring of mutant BCR/ABL transcript levels using DNA sequencing from a qualitative level to a quantitative level.