Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with large heterogeneity at the clinical and molecular levels. As diagnostic procedures shift from bone marrow biopsies towards less invasive techniques, circulating small noncoding RNAs (sncRNAs) have become of particular interest as potential novel noninvasive biomarkers of the disease. We aimed to characterize the expression profiles of circulating sncRNAs of MDS patients and to search for specific RNAs applicable as potential biomarkers. We performed small RNA-seq in paired samples of total plasma and plasma-derived extracellular vesicles (EVs) obtained from 42 patients and 17 healthy controls and analyzed the data with respect to the stage of the disease, patient survival, response to azacitidine, mutational status, and RNA editing. Significantly higher amounts of RNA material and a striking imbalance in RNA content between plasma and EVs (more than 400 significantly deregulated sncRNAs) were found in MDS patients compared to healthy controls. Moreover, the RNA content of EV cargo was more homogeneous than that of total plasma, and different RNAs were deregulated in these two types of material. Differential expression analyses identified that many hematopoiesis-related miRNAs (e.g., miR-34a, miR-125a, and miR-150) were significantly increased in MDS and that miRNAs clustered on 14q32 were specifically increased in early MDS. Only low numbers of circulating sncRNAs were significantly associated with somatic mutations in the SF3B1 or DNMT3A genes. Survival analysis defined a signature of four sncRNAs (miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p measured in EVs) as the most significantly associated with overall survival (HR = 5.866, p < 0.001). In total plasma, we identified five circulating miRNAs (miR-423-5p, miR-126-3p, miR-151a-3p, miR-125a-5p, and miR-199a-3p) whose combined expression levels could predict the response to azacitidine treatment. In conclusion, our data demonstrate that circulating sncRNAs show specific patterns in MDS and that their expression changes during disease progression, providing a rationale for the potential clinical usefulness of circulating sncRNAs in MDS prognosis. However, monitoring sncRNA levels in total plasma or in the EV fraction does not reflect one another, instead, they seem to represent distinctive snapshots of the disease and the data should be interpreted circumspectly with respect to the type of material analyzed.

The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the −11.7 kb and −0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34− cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.

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.

Background: myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder with an incompletely known pathogenesis. Long noncoding RNAs (lncRNAs) play multiple roles in hematopoiesis and represent a new class of biomarkers and therapeutic targets, but information on their roles in MDS is limited. Aims: here, we aimed to characterize lncRNAs deregulated in MDS that may function in disease pathogenesis. In particular, we focused on the identification of lncRNAs that could serve as novel potential biomarkers of adverse outcomes in MDS. Methods: we performed microarray expression profiling of lncRNAs and protein-coding genes (PCGs) in the CD34+ bone marrow cells of MDS patients. Expression profiles were analyzed in relation to different aspects of the disease (i.e., diagnosis, disease subtypes, cytogenetic and mutational aberrations, and risk of progression). LncRNA-PCG networks were constructed to link deregulated lncRNAs with regulatory mechanisms associated with MDS. Results: we found several lncRNAs strongly associated with disease pathogenesis (e.g., H19, WT1-AS, TCL6, LEF1-AS1, EPB41L4A-AS1, PVT1, GAS5, and ZFAS1). Of these, downregulation of LEF1-AS1 and TCL6 and upregulation of H19 and WT1-AS were associated with adverse outcomes in MDS patients. Multivariate analysis revealed that the predominant variables predictive of survival are blast count, H19 level, and TP53 mutation. Coexpression network data suggested that prognosis-related lncRNAs are predominantly related to cell adhesion and differentiation processes (H19 and WT1-AS) and mechanisms such as chromatin modification, cytokine response, and cell proliferation and death (LEF1-AS1 and TCL6). In addition, we observed that transcriptional regulation in the H19/IGF2 region is disrupted in higher-risk MDS, and discordant expression in this locus is associated with worse outcomes. Conclusions: we identified specific lncRNAs contributing to MDS pathogenesis and proposed cellular processes associated with these transcripts. Of the lncRNAs associated with patient prognosis, the level of H19 transcript might serve as a robust marker comparable to the clinical variables currently used for patient stratification.

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.