Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.

Extracellular vesicles have created great interest as possible source of biomarkers for different biological processes and diseases. Although the biological function of these vesicles is not fully understood, it is clear that they participate in the removal of unnecessary cellular material and act as carriers of various macromolecules and signals between the cells. In this report, we analyzed the proteome of extracellular vesicles secreted by primary hepatocytes. We used one- and two-dimensional liquid chromatography combined with data-independent mass spectrometry. Employing label-free quantitative proteomics, we detected significant changes in vesicle protein expression levels in this in vitro model after exposure to well-known liver toxins (galactosamine and Escherichia coli-derived lipopolysaccharide). The results allowed us to identify candidate markers for liver injury. We validated a number of these markers in vivo, providing the basis for the development of novel methods to evaluate drug toxicity. This report strongly supports the application of proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems. Analysis of such systems should help to identify specific markers for various biological processes and pathological conditions.

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies.

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.

Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are chronic myeloid clonal neoplasms. To date, the only potentially curative therapy for these disorders remains allogeneic hematopoietic progenitor cell transplantation (HCT), although patient eligibility is limited due to high morbimortality associated with this procedure coupled with advanced age of most patients. Dopamine receptors (DRs) and serotonin receptors type 1 (HTR1s) were identified as cancer stem cell therapeutic targets in acute myeloid leukemia. Given their close pathophysiologic relationship, expression of HTR1s and DRs was interrogated in MDS and CMML. Both receptors were differentially expressed in patient samples compared to healthy donors. Treatment with HTR1B antagonists reduced cell viability. HTR1 antagonists showed a synergistic cytotoxic effect with currently approved hypomethylating agents in AML cells. Our results suggest that HTR1B constitutes a novel therapeutic target for MDS and CMML. Due to its druggability, the clinical development of new regimens based on this target is promising.

There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of COMT activity in extracellular vesicles secreted by hepatocytes supporting a potential role of these vesicles in catecholamines and catecholestrogens metabolisms. In addition, the presence of COMT activity in extracellular vesicles opens new possibilities to develop tools to evaluate personalized drug response in a low invasive manner.

Hepatocytes release extracellular vesicles (EVs) loaded with signaling molecules and enzymes into the bloodstream. Although the importance of EVs in the intercellular communication is already recognized, the metabolic impact of the enzymes carried by these vesicles is still unclear. We evaluated the global effect of the enzymatic activities of EVs by performing untargeted metabolomic profiling of serum samples after their exposure to EVs. This approach revealed a significant change in the abundance of 94 serum metabolic signals. Our study shows that these vesicles modify the concentration of metabolites of different chemical nature including metabolites related to arginine metabolism, which regulates vascular function. To assess the functional relevance of this finding, we examined the levels of arginase-1 protein and its activity in the hepatic EVs carrying the exosomal markers CD81 and CD63. Remarkably, the arginase activity was also detected in EVs isolated from the serum in vivo, and this vesicular activity significantly increased under liver-damaging conditions. Finally, we demonstrated that EVs secreted by hepatocytes inhibited the acetylcholine-induced relaxation in isolated pulmonary arteries, via an arginase-dependent mechanism. In summary, our study demonstrates that the hepatocyte-released EVs are metabolically active, affecting a number of serum metabolites involved in oxidative stress metabolism and the endothelial function.

Drug-induced liver injury (DILI) is a serious worldwide health problem that accounts for more than 50% of acute liver failure. There is a great interest in clinical diagnosis and pharmaceutical industry to elucidate underlying molecular mechanisms and find noninvasive biomarkers for this pathology. Cell-secreted extracellular vesicles (EVs) have provided a new biological source to identify low disease invasive markers. Despite the intense research developed on these vesicles, there is currently a gap on their patho-physiological effects. Here, we study EVs secreted by primary rat hepatocytes challenged with galactatosamine (GalN), acetaminophen, or diclofenac as DILI in vitromodels. Proteomics analysis of these EVs revealed an increase in enzymes already associated with liver damage, such as catecholamine-methyl transferase and arginase 1. An increase in translation-related proteins and a decrease in regulators of apoptosis were also observed. In addition, we show the presence of enzymatic activity of P450 cytochrome 2d1 in EVs. The activity specifically is decreased in EVs secreted by hepatocytes after acetaminophen treatment and increased in EVs derived from GalN-treated hepatocytes. By using in vivo preclinical models, we demonstrate the presence of this cytochrome activity in circulation under normal conditions and an increased activity after GalN-induced injury. Conclusion: Hepatocyte-secreted EVs carry active xenobiotic-metabolizing enzymes that might be relevant in extracellular metabolism of drugs and be associated with DILI. (Hepatology Communications 2018;0:00-00).

Myelodysplastic syndromes (MDS) are stem cell disorders caused by various gene abnormalities. We performed targeted deep sequencing in 39 patients with high-risk MDS and secondary acute myeloid leukemia (sAML) at diagnosis and follow-up (response and/or relapse), with the aim to define their mutational status, to establish if specific mutations are biomarkers of response to 5-azacytidine (AZA) and/or may have impact on survival. Overall, 95% of patients harbored at least one mutation. TP53, DNMT3A and SRSF2 were the most frequently altered genes. Mutations in TP53 correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS) in univariate analysis. Patients with SRSF2 mutations were associated with better OS and PFS. Response rate was 55%; but we could not correlate the presence of TET2 and TP53 mutations with AZA response. Patients with sAML presented more variations than patients with high-risk MDS, and usually at relapse the number of mutations increased, supporting the idea that in advanced stages of the disease there is a greater genomic complexity. These results confirm that mutation analysis can add prognostic value to high-risk MDS and sAML patients, not only at diagnosis but also at follow-up.

Extracellular vesicles (EV) are emerging structures with promising properties for intercellular communication. In addition, the characterization of EV in biofluids is an attractive source of non-invasive diagnostic, prognostic and predictive biomarkers. Here we show that urinary EV (uEV) from prostate cancer (PCa) patients exhibit genuine and differential physical and biological properties compared to benign prostate hyperplasia (BPH). Importantly, transcriptomics characterization of uEVs led us to define the decreased abundance of Cadherin 3, type 1 (CDH3) transcript in uEV from PCa patients. Tissue and cell line analysis strongly suggested that the status of CDH3 in uEVs is a distal reflection of changes in the expression of this cadherin in the prostate tumor. CDH3 was negatively regulated at the genomic, transcriptional, and epigenetic level in PCa. Our results reveal that uEVs could represent a non-invasive tool to inform about the molecular alterations in PCa.

Liver constitutes the major metabolic factory in the organism and is involved in the synthesis, secretion and clearance of many blood-circulating molecules. Previously, we have characterised the protein and RNA cargo of extracellular vesicles (EVs) secreted by two hepatic cellular models, a mouse hepatocyte progenitor cell line (MLP29) and primary rat hepatocytes (RHs). Here, we report the metabolome profile of these vesicles by performing a targeted UHPLC-MS metabolomics analysis of these two cellular models and their respective secreted EVs. Visual inspection of the data through principal component analysis allows clear separation of the metabolic profile of cells and EVs, and also of both cellular models. Correlation matrix supported that lipid composition of EVs is mainly determined by parent cell composition. EVs derived from MLP29 and RHs showed a negative correlation in their percentage composition of ceramides, glycerophospholipids, sphingomyelins and triglycerides. Metabolites enriched in EVs were also different depending on the cellular model. EVs secreted by MLP29 were enriched in different species of sphingomyelins and ceramides underrepresented in EVs secreted by RHs. Remarkably, triglycerides constitute an important percentage of the composition of EVs derived from RHs. We further investigate if the differences in lipid composition were also accompanied by differences in mechanical behaviour, by using atomic force microscopy complemented with nanoindentation-based methodology. To compare the stiffness and brittleness of EVs derived from MLP29 cell line and RH primary cells, FZ curves were performed in the centre of single vesicles and the differences found in their force-vs.-indentation curves suggest that RHs EVs are softer (less stiff) and less resistance to mechanical failure than MLP29 EVs. Therefore, we can conclude that EVs from different origin carry a characteristic lipid composition related to their parental cell composition, and exhibit different mechanical properties. Abbreviations: For the identification of the different species of lipids, the following abbreviations has been employed: Cer, ceramide; ChoE, Cholesteryl Ester; CMH, monohexosylceramide; DAG, diglycerid; LPC, lysophosphatidylcholin; LPI, lysophosphatidyinositol; PC, phosphocoline; PE, phoethanolamine; PI, phosphoinositol; SM, sphingomyelin; TAG, triglycerid.

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) are key components of the hematopoietic niche thought to have a direct role in leukemia pathogenesis. BM-MSCs from patients with acute myeloid leukemia (AML) have been poorly characterized due to disease heterogeneity. We report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR), intermediate-risk (IR), and high-risk (HR) subgroups. Stable MSC cultures were successfully established and characterized from 40 of 46 AML patients irrespective of the risk subgroup. AML-derived BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, but displayed higher clonogenic potential than healthy donor (HD)-derived BM-MSCs. Although HD- and AML-derived BM-MSCs equally provided chemoprotection to AML cells in vitro, AML-derived BM-MSCs were more immunosuppressive/anti-inflammatory, enhanced suppression of lymphocyte proliferation, and diminished secretion of pro-inflammatory cytokines. Multivariate analysis revealed that the level of interleukin-10 produced by AML-derived BM-MSCs as an independent prognostic factor negatively affected overall survival. Collectively our data show that AML-derived BM-MSCs are not tumor related, but display functional differences contributing to therapy resistance and disease evolution.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has emerged as a powerful tool to decipher clonal architecture and to further understand cancer and other diseases at higher resolution level compared to bulk sequencing techniques. In this study, a SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available follow-up samples. Selected somatic alterations were further analyzed in SC by high-throughput qPCR (Biomark HD, Fluidigm) using specific TaqMan assays. A median of 175 cells per sample were analyzed. Inferred clonal architectures were relatively simple and either linear or branching. Similar to previous studies based on bulk sequencing to infer clonal architecture, we were able to observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific molecular characterization.

The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

Alterations in epigenetic marks, such as DNA methylation, represent a hallmark of cancer that has been successfully exploited for therapy in myeloid malignancies. Hypomethylating agents (HMA), such as azacitidine, have become standard-of-care therapy to treat myelodysplastic syndromes (MDS), myeloid neoplasms that can evolve into acute myeloid leukemia. However, our capacity to identify who will respond to HMAs, and the duration of response, remains limited. To shed light on this question, we have leveraged the unprecedented analytic power of single-cell technologies to simultaneously map the genome and immunoproteome of MDS samples throughout clinical evolution. We were able to chart the architecture and evolution of molecular clones in precious paired bone marrow MDS samples at diagnosis and posttreatment to show that a combined imbalance of specific cell lineages with diverse mutational profiles is associated with the clinical response of patients with MDS to hypomethylating therapy.