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Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ([almost equal to]22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

Key Points FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase (RTK) involved in the proliferation, differentiation and apoptosis of haematopoietic cells. It is mainly expressed by early myeloid and lymphoid progenitor cells. Many cells of the haematopoietic system produce FLT3 ligand (FLT3L), which promotes dimerization and activation of FLT3. The activated receptor then activates the phosphatidylinositol 3-kinase (PI3K) and RAS signal-transduction cascades. The FLT3 internal tandem duplication (ITD) results from a head-to-tail duplication of 3–400 base pairs in exons 14 or 15, which encode the juxtamembrane domain of FLT3. Point mutations in FLT3 occur in heavily conserved areas of the intracellular tyrosine-kinase domain (TKD), homologous to point mutations that are seen in other RTKs such as KIT and FMS. FLT3 mutations are the most frequent genetic lesion seen in acute myeloid leukaemia (AML). The prevalence of FLT3 ITDs is 15–35%, with an additional 5–10% of patients having FLT3 TKD mutations. Both types of FLT3 mutation cause ligand-independent activation of the receptor and activation of downstream signalling pathways. The presence of a FLT3 ITD is associated with poor clinical outcome in both paediatric and adult patients with AML. Several drugs that target FLT3 are in early clinical trials. Normal haematopoietic cells use complex systems to control proliferation, differentiation and cell death. The control of proliferation is, in part, accomplished through the ligand-induced stimulation of receptor tyrosine kinases, which signal to downstream effectors through the RAS pathway. Recently, mutations in the FMS-like tyrosine kinase 3 ( FLT3 ) gene, which encodes a receptor tyrosine kinase, have been found to be the most common genetic lesion in acute myeloid leukaemia (AML), occurring in ∼25% of cases. Exploring the mechanism by which these FLT3 mutations cause uncontrolled proliferation might lead to a better understanding of how cells become cancerous and provide insights for the development of new drugs.

Substantial effort is currently devoted to identifying cancer-associated alterations using genomics. Here, we show that standard blood collection procedures rapidly change the transcriptional and posttranscriptional landscapes of hematopoietic cells, resulting in biased activation of specific biological pathways; up-regulation of pseudogenes, antisense RNAs, and unannotated coding isoforms; and RNA surveillance inhibition. Affected genes include common mutational targets and thousands of other genes participating in processes such as chromatin modification, RNA splicing, T- and B-cell activation, and NF-κB signaling. The majority of published leukemic transcriptomes exhibit signals of this incubation-induced dysregulation, explaining up to 40% of differences in gene expression and alternative splicing between leukemias and reference normal transcriptomes. The effects of sample processing are particularly evident in pan-cancer analyses. We provide biomarkers that detect prolonged incubation of individual samples and show that keeping blood on ice markedly reduces changes to the transcriptome. In addition to highlighting the potentially confounding effects of technical artifacts in cancer genomics data, our study emphasizes the need to survey the diversity of normal as well as neoplastic cells when characterizing tumors. Significance An important goal of cancer biology is to identify molecular differences between normal and cancer cells. Accordingly, many large-scale initiatives to characterize both solid and liquid tumor samples with genomics technologies are currently underway. Here, we show that standard blood collection procedures cause rapid changes to the transcriptomes of hematopoietic cells. The resulting transcriptional and posttranscriptional artifacts are visible in most published leukemia genomics datasets and hinder the identification and interpretation of cancer-specific alterations.

In acute myeloid leukemia (AML) and blast crisis (BC) chronic myeloid leukemia (CML) normal differentiation is impaired. Differentiation of immature stem/progenitor cells is critical for normal blood cell function. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Aberrant miRNA expression is a feature of leukemia and miRNAs also play a significant role in normal hematopoiesis and differentiation. We have identified miRNAs differentially expressed in AML and BC CML and identified a new role for miR-150 in myeloid differentiation. Expression of miR-150 is low or absent in BC CML and AML patient samples and cell lines. We have found that expression of miR-150 in AML cell lines, CD34+ progenitor cells from healthy individuals, and primary BC CML and AML patient samples at levels similar to miR-150 expression in normal bone marrow promotes myeloid differentiation of these cells. MYB is a direct target of miR-150, and we have identified that the observed phenotype is partially mediated by MYB. In AML cell lines, differentiation of miR-150 expressing cells occurs independently of retinoic acid receptor α (RARA) signaling. High-throughput gene expression profiling (GEP) studies of the AML cell lines HL60, PL21, and THP-1 suggest that activation of CEPBA, CEBPE, and cytokines associated with myeloid differentiation in miR-150 expressing cells as compared to control cells contributes to myeloid differentiation. These data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role for this miRNA, and that absent or low miR-150 expression contributes to blocked myeloid differentiation in acute leukemia cells.

The t(8;21) and Inv(16) translocations disrupt the normal function of core binding factors alpha (CBFA) and beta (CBFB), respectively. These translocations represent two of the most common genomic abnormalities in acute myeloid leukemia (AML) patients, occurring in approximately 25% pediatric and 15% of adult with this malignancy. Both translocations are associated with favorable clinical outcomes after intensive chemotherapy, and given the perceived mechanistic similarities, patients with these translocations are frequently referred to as having CBF-AML. It remains uncertain as to whether, collectively, these translocations are mechanistically the same or impact different pathways in subtle ways that have both biological and clinical significance. Therefore, we used transcriptome sequencing (RNA-seq) to investigate the similarities and differences in genes and pathways between these subtypes of pediatric AMLs. Diagnostic RNA from patients with t(8;21) (N = 17), Inv(16) (N = 14), and normal karyotype (NK, N = 33) were subjected to RNA-seq. Analyses compared the transcriptomes across these three cytogenetic subtypes, using the NK cohort as the control. A total of 1291 genes in t(8;21) and 474 genes in Inv(16) were differentially expressed relative to the NK controls, with 198 genes differentially expressed in both subtypes. The majority of these genes (175/198; binomial test p-value < 10(-30)) are consistent in expression changes among the two subtypes suggesting the expression profiles are more similar between the CBF cohorts than in the NK cohort. Our analysis also revealed alternative splicing events (ASEs) differentially expressed across subtypes, with 337 t(8;21)-specific and 407 Inv(16)-specific ASEs detected, the majority of which were acetylated proteins (p = 1.5 x 10(-51) and p = 1.8 x 10(-54) for the two subsets). In addition to known fusions, we identified and verified 16 de novo fusions in 43 patients, including three fusions involving NUP98 in six patients. Clustering of differentially expressed genes indicated that the homeobox (HOX) gene family, including two transcription factors (MEIS1 and NKX2-3) were down-regulated in CBF compared to NK samples. This finding supports existing data that the dysregulation of HOX genes play a central role in biology CBF-AML hematopoiesis. These data provide comprehensive transcriptome profiling of CBF-AML and delineate genes and pathways that are differentially expressed, providing insights into the shared biology as well as differences in the two CBF subsets.

BackgroundMyelodysplastic syndromes (MDS) arise from somatic mutations acquired in hematopoietic stem and progenitor cells, causing cytopenias and predisposing to transformation into secondary acute myeloid leukemia (sAML). Recurrent mutations in spliceosome genes, including U2AF1, are attractive therapeutic targets as they are prevalent in MDS and sAML, arise early in neoplastic cells, and are generally absent from normal cells, including normal hematopoietic cells. MDS and sAML are susceptible to T cell-mediated killing, and thus engineered T-cell immunotherapies hold promise for their treatment. We hypothesized that targeting spliceosome mutation-derived neoantigens with transgenic T-cell receptor (TCR) T cells would selectively eradicate malignant cells in MDS and sAML.MethodsWe identified candidate neoantigen epitopes from recurrent protein-coding mutations in the spliceosome genes SRSF2 and U2AF1 using a multistep in silico process. Candidate epitopes predicted to bind human leukocyte antigen (HLA) class I, be processed and presented from the parent protein, and not to be subject to tolerance then underwent in vitro immunogenicity screening. CD8+ T cells recognizing immunogenic neoantigen epitopes were evaluated in in vitro assays to assess functional avidity, confirm the predicted HLA restriction, the potential for recognition of similar peptides, and the ability to kill neoplastic cells in an antigen-specific manner. Neoantigen-specific TCR were sequenced, cloned into lentiviral vectors, and transduced into third-party T cells after knock-out of endogenous TCR, then tested in vitro for specificity and ability to kill neoplastic myeloid cells presenting the neoantigen. The efficacy of neoantigen-specific T cells was evaluated in vivo in a murine cell line-derived xenograft model.ResultsWe identified two neoantigens created from a recurrent mutation in U2AF1, isolated CD8+ T cells specific for the neoantigens, and demonstrated that transferring their TCR to third-party CD8+ T cells is feasible and confers specificity for the U2AF1 neoantigens. Finally, we showed that these neoantigen-specific TCR-T cells do not recognize normal hematopoietic cells but efficiently kill malignant myeloid cells bearing the specific U2AF1 mutation, including primary cells, in vitro and in vivo.ConclusionsThese data serve as proof-of-concept for developing precision medicine approaches that use neoantigen-directed T-cell receptor-transduced T cells to treat MDS and sAML.

TNFα levels are elevated in the marrows of patients with myelodysplastic syndrome (MDS) and are associated with high rates of apoptosis, which contributes to hematopoietic failure. We observed that exposure of human marrow stroma cell lines HS5 and HS27a to TNFα increases levels of IL-32 mRNA. IL-32, in turn, induces TNFα. Marrow stroma from patients with MDS expressed 14- to 17-fold higher levels of IL-32 mRNA than healthy controls. In contrast, cells from patients with chronic myelomonocytic leukemia (CMML) expressed only one tenth the level of IL-32 measured in healthy controls. Human KG1a leukemia cells underwent apoptosis when cocultured with HS5 stromal cells, but knockdown of IL-32 in the stromal cells by using siRNA abrogated apoptosis in the leukemia cells. IL-32 knockdown cells also showed dysregulation of VEGF and other cytokines. Furthermore, CD56⁺ natural killer cells from patients with MDS and CMML expressed IL-32 at lower levels than controls and exhibited reduced cytotoxic activity, which was unaffected by IL-2 treatment. We propose that IL-32 is a marrow stromal marker that distinguishes patients with MDS and CMML. Furthermore, IL-32 appears to contribute to the pathophysiology of MDS and may be a therapeutic target.

Background The recently updated European LeukemiaNet risk stratification guidelines combine cytogenetic abnormalities and genetic mutations to provide the means to triage patients with acute myeloid leukemia for optimal therapies. Despite the identification of many prognostic factors, relatively few have made their way into clinical practice. Methods In order to assess and improve the performance of the European LeukemiaNet guidelines, we developed novel prognostic models using the biomarkers from the guidelines, age, performance status and select transcript biomarkers. The models were developed separately for mononuclear cells and viable leukemic blasts from previously untreated acute myeloid leukemia patients (discovery cohort, N  = 185) who received intensive chemotherapy. Models were validated in an independent set of similarly treated patients (validation cohort, N  = 166). Results Models using European LeukemiaNet guidelines were significantly associated with clinical outcomes and, therefore, utilized as a baseline for comparisons. Models incorporating age and expression of select transcripts with biomarkers from European LeukemiaNet guidelines demonstrated higher area under the curve and C-statistics but did not show a substantial improvement in performance in the validation cohort. Subset analyses demonstrated that models using only the European LeukemiaNet guidelines were a better fit for younger patients (age < 55) than for older patients. Models integrating age and European LeukemiaNet guidelines visually showed more separation between risk groups in older patients. Models excluding results for ASXL1 , CEBPA , RUNX1 and TP53 , demonstrated that these mutations provide a limited overall contribution to risk stratification across the entire population, given the low frequency of mutations and confounding risk factors. Conclusions While European LeukemiaNet guidelines remain a critical tool for triaging patients with acute myeloid leukemia, the findings illustrate the need for additional prognostic factors, including age, to improve risk stratification.