BackgroundThrough high-throughput next-generation sequencing of promoters of solute carrier and ATP-binding cassette genes, which encode drug transporters, we aimed to identify SNPs associated with the response to imatinib administered for first-line treatment of patients with chronic myeloid leukemia.Methods
In silico analysis using publicly available databases was done to select the SLC and ABC genes and their promoters for the next-generation sequencing. SNPs associated with the imatinib response were identified using Fisher’s exact probability tests and subjected to the linkage disequilibrium analyses with regulatory loci of concerned genes. We analyzed cumulative achievement of major molecular response and probability of event free survival in relation to identified SNP genotypes in 129 CML patients and performed multivariate analysis for determination of genotypes as independent predictors of outcome. Gene expression analysis of eight cell lines naturally carrying different genotypes was performed to outline an impact of genotypes on the gene expression.ResultsWe observed significant differences in the frequencies of the rs460089-GC and rs460089-GG (SLC22A4) genotypes among rs2631365-TC (SLC22A5) genotype carriers that were associated with optimal and non-optimal responses, respectively. Loci rs460089 and rs2631365 were in highly significant linkage disequilibrium with 12 regulatory loci in introns of SLC22A4 and SLC22A5 encoding imatinib transporters. Genotype association analysis with the response to imatinib indicated that rs460089-GC carriers had a significantly higher probability of achieving a stable major molecular response (BCR-ABL1 transcript level below or equal to 0.1% in the international scale). In contrast, the rs460089-GG represented a risk factor for imatinib failure, which was significantly higher in rs460089-GG_rs2631365-TC carriers.ConclusionsThis exploratory study depicted potentially important genetic markers predicting outcome of imatinib treatment, which may be helpful for tailoring therapy in clinical practice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-017-0523-3) contains supplementary material, which is available to authorized users.
This work investigated patient-specific genomic BCR-ABL1 fusions as markers of measurable residual disease (MRD) in chronic myeloid leukemia, with a focus on relevance to treatment-free remission (TFR) after achievement of deep molecular response (DMR) on tyrosine kinase inhibitor (TKI) therapy.DNA and mRNA BCR-ABL1 measurements by qPCR were compared in 2189 samples (129 patients) and by digital PCR in 1279 sample (62 patients). A high correlation was found at levels of disease above MR4, but there was a poor correlation for samples during DMR. A combination of DNA and RNA MRD measurements resulted in a better prediction of molecular relapse-free survival (MRFS) after TKI stop (n=17) or scheduled interruption (n=25). At 18 months after treatment cessation, patients with stopped or interrupted TKI therapy who were DNA negative/RNA negative during DMR maintenance (green group) had a molecular relapse-free survival (MRFS) of 80% and 100%, respectively, compared to those who were DNA positive/RNA negative (MRFS= 57% and 67%, respectively; yellow group) or DNA positive/RNA positive (MRFS=20% for both cohorts; red group). Thus, we propose a "traffic light" stratification as a TFR predictor based on DNA and mRNA BCR-ABL1 measurements during DMR maintenance before TKI cessation.
Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR-ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR-ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib-resistant K562 (K562 IR ) cells. The K562 IR -derived exosomes were internalized by imatinib-sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance-associated markers using a deep label-free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon-induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562 IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562 IR exosomes, and also in the K562 IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.
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