Utilization of Next-Generation Deep Sequencing to Analyze BCR-ABL1 Kinase Domain Mutation for Imatinib-Resistant Chronic Myeloid Leukemia Patients in Vietnam

Background: Chronic myeloid leukemia is a clonal myeloproliferative neoplasm, characterized by the presence of chromosomal translocation t(9; 22)(q34; q11). This is found in over 95% of the cases and results in the BCR-ABL1 fusion protein with high tyrosine kinase activity. During the last decades, imatinib and other generations of tyrosine kinase inhibitor have been used effectively for target therapy of the disease. However, many of the drug resistant cases have been reported recently, due to the mutation within kinase domain of the BCR-ABL1 fusion gene. In order to provide further information about this incidence, we performed a retrospective study of 141 imatinib-resistance chronic myeloid leukemia patients to analyze kinase domain mutation by deep sequencing. Another group of 20 untreated patients were added as control. Methods: RNA from bone marrow cells were extracted and followed by cDNA synthesis. Nested polymerase chain reaction was performed to amplify kinase domain of the BCR-ABL1 fusion gene. The amplified products were monitored size, concentration and prepared DNA sequencing library. Sequence analysis was performed using Illumina MiSeq sequencer and Sequence Pilot software. The sequencing results were randomly chose for Sanger sequencing. Results: None of the control group was positive with kinase domain mutation. There were 47 out of 141 patients (33%) detected with at least one nucleotide substitution. The sequencing results were also confirmed by Sanger sequencing. Of those 47 samples, 72 nucleotide substitutions of 28 types, altered 24 codons were identified. Among those, Y253F/H, M351T, G250E, F359V/I and M244V were the most frequent mutations, while T315I took only 4.1%. There were also a number of samples harboring multiple substitutions and new variations. Conclusion: Nextgeneration deep sequencing is a sensitive and effective method to detect kinase domain mutation and our results could provide further information about the drug-resistance mutation in chronic myeloid leukemia.