The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability.

Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. Stem Cells 2019;37:1357-1368.

Deferasirox reduces oxidative DNA damage in bone marrow cells from myelodysplastic patients and improves their differentiation capacity.

Patients with low-risk myelodysplastic syndromes (MDS) usually develop iron overload. This leads to a high level of oxidative stress in the bone marrow (BM) and increases haematopoietic cell dysfunction. Our objective was to analyse whether chelation with deferasirox (DFX) alleviates the consequences of oxidative stress and improves BM cell functionality. We analysed 13 iron-overloaded MDS patients' samples before and 4-10 months after treatment with DFX. Using multiparametric flow cytometry analysis, we measured intracellular reactive oxygen species (ROS), DNA oxidation and double strand breaks. Haematopoietic differentiation capacity was analysed by colony-forming unit (CFU) assays. Compared to healthy donors, MDS showed a higher level of intracellular ROS and DNA oxidative damage in BM cells. DNA oxidative damage decreased following DFX treatment. Furthermore, the clonogenic assays carried out before treatment suggest an impaired haematopoietic differentiation. DFX seems to improve this capacity, as illustrated by a decreased cluster/CFU ratio, which reached values similar to controls. We conclude that BM cells from MDS are subject to higher oxidative stress conditions and show an impaired haematopoietic differentiation. These adverse features seem to be partially rectified after DFX treatment.

Multidimensional assessment of patient condition and mutational analysis in peripheral blood, as tools to improve outcome prediction in myelodysplastic syndromes: A prospective study of the Spanish MDS group.

The International Prognostic Scoring System and its revised form (IPSS-R) are the most widely used indices for prognostic assessment of patients with myelodysplastic syndromes (MDS), but can only partially account for the observed variation in patient outcomes. This study aimed to evaluate the relative contribution of patient condition and mutational status in peripheral blood when added to the IPSS-R, for estimating overall survival and the risk of leukemic transformation in patients with MDS. A prospective cohort (2006-2015) of 200 consecutive patients with MDS were included in the study series and categorized according to the IPSS-R. Patients were further stratified according to patient condition (assessed using the multidimensional Lee index for older adults) and genetic mutations (peripheral blood samples screened using next-generation sequencing). The change in likelihood-ratio was tested in Cox models after adding individual covariates. The addition of the Lee index to the IPSS-R significantly improved prediction of overall survival [hazard ratio (HR) 3.02, 95% confidence interval (CI) 1.96-4.66, P < 0.001), and mutational analysis significantly improved prediction of leukemic evolution (HR 2.64, 1.56-4.46, P < 0.001). Non-leukemic death was strongly linked to patient condition (HR 2.71, 1.72-4.25, P < 0.001), but not to IPSS-R score (P = 0.35) or mutational status (P = 0.75). Adjustment for exposure to disease-modifying therapy, evaluated as a time-dependent covariate, had no effect on the proposed model's predictive ability. In conclusion, patient condition, assessed by the multidimensional Lee index and patient mutational status can improve the prediction of clinical outcomes of patients with MDS already stratified by IPSS-R.

Insights into the human mesenchymal stromal/stem cell identity through integrative transcriptomic profiling.

BACKGROUND: Mesenchymal Stromal/Stem Cells (MSCs), isolated under the criteria established by the ISCT, still have a poorly characterized phenotype that is difficult to distinguish from similar cell populations. Although the field of transcriptomics and functional genomics has quickly grown in the last decade, a deep comparative analysis of human MSCs expression profiles in a meaningful cellular context has not been yet performed. There is also a need to find a well-defined MSCs gene-signature because many recent biomedical studies show that key cellular interaction processes (i.e. inmuno-modulation, cellular cross-talk, cellular maintenance, differentiation, epithelial-mesenchymal transition) are dependent on the mesenchymal stem cells within the stromal niche. RESULTS: In this work we define a core mesenchymal lineage signature of 489 genes based on a deep comparative analysis of multiple transcriptomic expression data series that comprise: (i) MSCs of different tissue origins; (ii) MSCs in different states of commitment; (iii) other related non-mesenchymal human cell types. The work integrates several public datasets, as well as de-novo produced microarray and RNA-Seq datasets. The results present tissue-specific signatures for adipose tissue, chorionic placenta, and bone marrow MSCs, as well as for dermal fibroblasts; providing a better definition of the relationship between fibroblasts and MSCs. Finally, novel CD marker patterns and cytokine-receptor profiles are unravelled, especially for BM-MSCs; with MCAM (CD146) revealed as a prevalent marker in this subtype of MSCs. CONCLUSIONS: The improved biomolecular characterization and the released genome-wide expression signatures of human MSCs provide a comprehensive new resource that can drive further functional studies and redesigned cell therapy applications.

Effect of mTORC1/mTORC2 inhibition on T cell function: potential role in graft-versus-host disease control.

The mechanistic target of rapamycin (mTOR) pathway is crucial for the activation and function of T cells, which play an essential role in the development of graft-versus-host disease (GvHD). Despite its partial ability to block mTOR pathway, the mTORC1 inhibitor rapamycin has shown encouraging results in the control of GvHD. Therefore, we considered that simultaneous targeting of both mTORC1 and mTORC2 complexes could exert a more potent inhibition of T cell activation and, thus, could have utility in GvHD control. To assess this assumption, we have used the dual mTORC1/mTORC2 inhibitors CC214-1 and CC214-2. In vitro studies confirmed the superior ability of CC214-1 versus rapamycin to block mTORC1 and mTORC2 activity and to reduce T cell proliferation. Both drugs induced a similar decrease in Th1/Th2 cytokine secretion, but CC214-1 was more efficient in inhibiting naïve T cell activation and the expression of T-cell activation markers. In addition, CC214-1 induced specific tolerance against alloantigens, while preserving anti-cytomegalovirus response. Finally, in a mouse model of GvHD, the administration of CC214-2 significantly improved mice survival and decreased GvHD-induced damages. In conclusion, the current study shows, for the first time, the immunosuppressive ability of CC214-1 on T lymphocytes and illustrates the role of CC214-2 in the allogeneic transplantation setting as a possible GvHD prophylaxis agent.

Comparative analysis of the immunomodulatory capacities of human bone marrow- and adipose tissue-derived mesenchymal stromal cells from the same donor.

BACKGROUND AIMS: The immunomodulatory properties of mesenchymal stromal cells (MSCs), together with their tissue regenerative potential, make them interesting candidates for clinical application. METHODS: In the current study, we analyzed the in vitro immunomodulatory effects of MSCs derived from bone marrow (BM-MSCs) and from adipose tissue (AT-MSCs) obtained from the same donor on both innate and acquired immunity cells. BM-MSCs and AT-MSCs were expanded to fourth or fifth passage and co-cultured with T cells, monocytes or natural killer (NK) cells isolated from human peripheral blood and stimulated in vitro. The possible differing impact of MSCs obtained from distinct sources on phenotype, cell proliferation and differentiation, cytokine production and function of these immune cells was comparatively analyzed. RESULTS: BM-MSCs and AT-MSCs induced a similar decrease in NK-cell proliferation, cytokine secretion and expression of both activating receptors and cytotoxic molecules. However, only BM-MSCs significantly reduced NK-cell cytotoxic activity, although both MSC populations showed the same susceptibility to NK-cell-mediated lysis. AT-MSCs were more potent in inhibiting dendritic-cell (DC) differentiation than BM-MSC, but both MSC populations similarly reduced the ability of DCs to induce CD4(+) T-cell proliferation and cytokine production. BM-MSCs and AT-MSCs induced a similar decrease in T-cell proliferation and production of inflammatory cytokines after activation. CONCLUSIONS: AT-MSCs and BM-MSCs from the same donor had similar immunomodulatory capacity on both innate and acquired immunity cells. Thus, other variables, such as accessibility of samples or the frequency of MSCs in the tissue should be considered to select the source of MSC for cell therapy.

MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry.

BACKGROUND: Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer. METHODS: Human MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed. RESULTS: EV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 µm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP. Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively. CONCLUSION: We described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility.

Immunomodulatory effects of bone marrow versus adipose tissue-derived mesenchymal stromal cells on NK cells: implications in the transplantation setting.

INTRODUCTION: The ability of mesenchymal stromal cells (MSC) to suppress T-cell function has prompted their therapeutic use for graft-versus-host disease (GVHD) control. However, as MSC also modulate the activity of NK cells, which play an important role in graft-versus-leukemia (GVL) reaction, their administration could hamper this beneficial effect of allogeneic hematopoietic stem cell transplantation. MSC can be expanded from several sources, especially bone marrow and fat, but it is not well established if the cell source makes a difference in their immunoregulatory capacity. OBJECTIVE: The aim of this study was to compare the immunomodulatory effect of MSC derived from bone marrow (BM-CSM) or adipose tissue (AT-MSC) on NK cells, to determine whether the use of MSC from one or the other origin could be more favorable to preserve NK cell activity and, therefore, GVL. METHODS: Human NK cells were stimulated with IL-15 in the presence of BM-MSC or AT-MSC. The effect of both MSC populations on NK cell proliferation, cell cycle progression, and CD56 expression was analyzed by flow cytometry. Cytokine secretion was measured by ELISA, and cytotoxic activity was assessed by calcein release assays. RESULTS: Although both BM-MSC and AT-MSC induced a similar inhibition of NK cell proliferation, only BM-MSC decreased significantly NK cell cytotoxic activity and showed a trend for a higher reduction of IFN-γ secretion. CONCLUSION: These results suggest that, in the context of GVHD inhibition, the use of AT-MSC rather than BM-MSC could further preserve NK cell activity and, thus, favor GVL.

Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European LeukemiaNet.

Within the myelodysplastic syndrome (MDS) work package of the European LeukemiaNet, an Expert Panel was selected according to the framework elements of the National Institutes of Health Consensus Development Program. A systematic review of the literature was performed that included indexed original papers, indexed reviews and educational papers, and abstracts of conference proceedings. Guidelines were developed on the basis of a list of patient- and therapy-oriented questions, and recommendations were formulated and ranked according to the supporting level of evidence. MDSs should be classified according to the 2008 World Health Organization criteria. An accurate risk assessment requires the evaluation of not only disease-related factors but also of those related to extrahematologic comorbidity. The assessment of individual risk enables the identification of fit patients with a poor prognosis who are candidates for up-front intensive treatments, primarily allogeneic stem cell transplantation. A high proportion of MDS patients are not eligible for potentially curative treatment because of advanced age and/or clinically relevant comorbidities and poor performance status. In these patients, the therapeutic intervention is aimed at preventing cytopenia-related morbidity and preserving quality of life. A number of new agents are being developed for which the available evidence is not sufficient to recommend routine use. The inclusion of patients into prospective clinical trials is strongly recommended.

Transcriptomic portrait of human Mesenchymal Stromal/Stem Cells isolated from bone marrow and placenta.

BACKGROUND: Human Mesenchymal Stromal/Stem Cells (MSCs) are adult multipotent cells that behave in a highly plastic manner, inhabiting the stroma of several tissues. The potential utility of MSCs is nowadays strongly investigated in the field of regenerative medicine and cell therapy, although many questions about their molecular identity remain uncertain. RESULTS: MSC primary cultures from human bone marrow (BM) and placenta (PL) were derived and verified by their immunophenotype standard pattern and trilineage differentiation potential. Then, a broad characterization of the transcriptome of these MSCs was performed using RNA deep sequencing (RNA-Seq). Quantitative analysis of these data rendered an extensive expression footprint that includes 5,271 protein-coding genes. Flow cytometry assays of canonical MSC CD-markers were congruent with their expression levels detected by the RNA-Seq. Expression of other recently proposed MSC markers (CD146, Nestin and CD271) was tested in the placenta samples, finding only CD146 and Nestin. Functional analysis revealed enrichment in stem cell related genes and mesenchymal regulatory transcription factors (TFs). Analysis of TF binding sites (TFBSs) identified 11 meta-regulators, including factors KLF4 and MYC among them. Epigenetically, hypomethylated promoter patterns supported the active expression of the MSC TFs found. An interaction network of these TFs was built to show up their links and relations. Assessment of dissimilarities between cell origins (BM versus PL) disclosed two hundred differentially expressed genes enrolled in microenvironment processes related to the cellular niche, as regulation of bone formation and blood vessel morphogenesis for the case of BM-MSCs. By contrast genes overexpressed in PL-MSCs showed functional enrichment on mitosis, negative regulation of cell-death and embryonic morphogenesis that supported the higher growth rates observed in the cultures of these fetal cells and their closer links with development processes. CONCLUSIONS: The results present a transcriptomic portrait of the human MSCs isolated from bone marrow and placenta. The data are released as a cell-specific resource, providing a comprehensive expression footprint of the MSCs useful to better understand their cellular and molecular biology and for further investigations on the isolation and biomedical use of these multipotent cells.

Multivariate time-dependent comparison of the impact of lenalidomide in lower-risk myelodysplastic syndromes with chromosome 5q deletion.

The impact of lenalidomide treatment on long-term outcomes of patients with lower risk myelodysplastic syndromes (MDS) and chromosome 5q deletion (del(5q)) is unclear. This study used time-dependent multivariate methodology to analyse the influence of lenalidomide therapy on overall survival (OS) and acute myeloblastic leukaemia (AML) progression in 215 patients with International Prognostic Scoring System (IPSS) low or intermediate-1 risk and del(5q). There were significant differences in several relevant characteristics at presentation between patients receiving (n = 86) or not receiving lenalidomide (n = 129). The 5-year time-dependent probabilities of OS and progression to AML were 62% and 31% for patients receiving lenalidomide and 42% and 25% for patients not receiving lenalidomide; differences were not statistically significant in multivariate analysis that included all variables independently associated with those outcomes (OS, P = 0·45; risk of AML, P = 0·31, respectively). Achievement of RBC transfusion independency (P = 0·069) or cytogenetic response (P = 0·021) after lenalidomide was associated with longer OS in multivariate analysis. These data clearly show that response to lenalidomide results in a substantial clinical benefit in lower risk MDS patients with del(5q). Lenalidomide treatment does not appear to increase AML risk in this population of patients.

Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease.

Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral pancytopenia and marrow hypoplasia. The majority of cases of aplastic anemia remain idiopathic, although hematopoietic stem cell deficiency and impaired immune responses are hallmarks underlying the bone marrow failure in this condition. Mesenchymal stem/stromal cells constitute an essential component of the bone marrow hematopoietic microenvironment because of their immunomodulatory properties and their ability to support hematopoiesis, and they have been involved in the pathogenesis of several hematologic malignancies. We investigated whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the pathogenesis of aplastic anemia. We found that mesenchymal stem cell cultures can be established from the bone marrow of aplastic anemia patients and display the same phenotype and differentiation potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34(+) cells, and maintain their immunosuppressive and anti-inflammatory properties. These data demonstrate that bone marrow mesenchymal stem cells from patients with aplastic anemia do not have impaired functional and immunological properties, suggesting that they do not contribute to the pathogenesis of the disease.

Outcomes in RBC transfusion-dependent patients with Low-/Intermediate-1-risk myelodysplastic syndromes with isolated deletion 5q treated with lenalidomide: a subset analysis from the MDS-004 study.

OBJECTIVE: A subset analysis of the randomised, phase 3, MDS-004 study to evaluate outcomes in patients with International Prognostic Scoring System (IPSS)-defined Low-/Intermediate (Int)-1-risk myelodysplastic syndromes (MDS) with isolated del(5q). METHODS: Patients received lenalidomide 10 mg/d (days 1-21; n = 47) or 5 mg/d (days 1-28; n = 43) on 28-d cycles or placebo (n = 45). From the placebo and lenalidomide 5 mg groups, 84% and 58% of patients, respectively, crossed over to lenalidomide 5 or 10 mg at 16 wk, respectively. RESULTS: Rates of red blood cell-transfusion independence (RBC-TI) ≥182 d were higher in the lenalidomide 10 mg (57.4%; P < 0.0001) and 5 mg (37.2%; P = 0.0001) groups vs. placebo (2.2%). Cytogenetic response rates (major + minor responses) were 56.8% (P < 0.0001), 23.1% (P = 0.0299) and 0%, respectively. Two-year cumulative risk of acute myeloid leukaemia progression was 12.6%, 17.4% and 16.7% in the lenalidomide 10 mg, 5 mg, and placebo groups, respectively. In a 6-month landmark analysis, overall survival was longer in lenalidomide-treated patients with RBC-TI ≥182 d vs. non-responders (P = 0.0072). The most common grade 3-4 adverse event was myelosuppression. CONCLUSIONS: These data support the clinical benefits and acceptable safety profile of lenalidomide in transfusion-dependent patients with IPSS-defined Low-/Int-1-risk MDS with isolated del(5q).

Low dose lenalidomide versus placebo in non-transfusion dependent patients with low risk, del(5q) myelodysplastic syndromes (SintraREV): a randomised, double-blind, phase 3 trial.

BACKGROUND: Lenalidomide is the standard of care for patients who are transfusion dependent with chromosome 5q deletion (del[5q]) myelodysplastic syndromes. In the SintraREV trial, we aimed to investigate whether an early intervention of low lenalidomide doses for 2 years could delay transfusion dependency in patients with anaemia who were not transfusion dependent. METHODS: This randomised, double-blind, phase 3 trial, was conducted at 22 sites (University Hospitals) in Spain, France, and Germany. Eligible patients were aged 18 years or older diagnosed with low-risk or intermediate-1-risk del(5q) myelodysplastic syndromes with non-transfusion-dependent anaemia (according to the IPSS), were erythropoietin-stimulating agents naive, and had an ECOG performance status of 2 or less. Patients were randomly assigned (2:1) by means of a telephone system to receive lenalidomide 5 mg daily in 28-day cycles versus placebo for 2 years. The primary endpoint was time to transfusion dependency based on blinded independent central review. Analysis were by intent-to-treat (ITT) and evaluable population. Safety analyses included all participants who received at least one dose of treatment. This trial is registered with ClinicalTrials.gov (NCT01243476) and EudraCT (2009-013619-36) and is complete. FINDINGS: Between Feb 15, 2010, and Feb 21, 2018, 61 patients were randomly assigned to receive lenalidomide (n=40; two did not receive treatment) or placebo (n=21). The median age was 72·2 (IQR 65·4-81·9) years, 50 (82%) patients were female, and 11 (18%) were male. The median follow-up time was 60·6 (IQR 32·1-73·9) months. Regarding primary endpoint, median time to transfusion dependency was not reached (95% CI not applicable) in the lenalidomide group versus 11·6 months (95% CI 0·00-30·11) in the placebo group (p=0·0027). Lenalidomide significantly reduced the risk of transfusion dependency by 69·8% (hazard ratio 0·302, 95% CI 0·132-0·692; p=0·0046). The most frequent treatment-related adverse event was neutropenia, occurring in 24 (63%) of 38 patients in the lenalidomide group (grade 3 and 4 in 17 [45%] patients and one [3%], respectively) and in four (19%) of 21 patients in the placebo group (grade 3 in one [5%] patient). Thrombocytopenia was detected in seven (18%) of 38 patients receiving lenalidomide (grade 3 in two [5%] patients). Regarding the non-haematological toxicity, skin disorders (rash nine [23%] of 38 patients) were the most frequently described toxicities among patients receiving lenalidomide, being grade 3 in one (3%) of 38 patients. 19 serious adverse events were reported in 13 patients, 18 in the lenalidomide group and one in the placebo group, five of which were potentially related to the study drug. No treatment-related deaths were identified. INTERPRETATION: An early approach with low doses of lenalidomide across two years delays the time to transfusion dependency and improves the rate and quality of the responses, with a manageable safety profile in patients who are non-transfusion dependent with del(5q) low-risk myelodysplastic syndromes. FUNDING: Bristol Myers Squibb.

A randomized phase 3 study of lenalidomide versus placebo in RBC transfusion-dependent patients with Low-/Intermediate-1-risk myelodysplastic syndromes with del5q.

This phase 3, randomized, double-blind study assessed the efficacy and safety of lenalidomide in 205 red blood cell (RBC) transfusion-dependent patients with International Prognostic Scoring System Low-/Intermediate-1-risk del5q31 myelodysplastic syndromes. Patients received lenalidomide 10 mg/day on days 1-21 (n = 69) or 5 mg/day on days 1-28 (n = 69) of 28-day cycles; or placebo (n = 67). Crossover to lenalidomide or higher dose was allowed after 16 weeks. More patients in the lenalidomide 10- and 5-mg groups achieved RBC-transfusion independence (TI) for ≥ 26 weeks (primary endpoint) versus placebo (56.1% and 42.6% vs 5.9%; both P < .001). Median duration of RBC-TI was not reached (median follow-up, 1.55 years), with 60% to 67% of responses ongoing in patients without progression to acute myeloid leukemia (AML). Cytogenetic response rates were 50.0% (10 mg) versus 25.0% (5 mg; P = .066). For the lenalidomide groups combined, 3-year overall survival and AML risk were 56.5% and 25.1%, respectively. RBC-TI for ≥ 8 weeks was associated with 47% and 42% reductions in the relative risks of death and AML progression or death, respectively (P = .021 and .048). The safety profile was consistent with previous reports. Lenalidomide is beneficial and has an acceptable safety profile in transfusion-dependent patients with Low-/Intermediate-1-risk del5q myelodysplastic syndrome. This trial was registered at www.clinicaltrials.gov as #NCT00179621.

Uptake and delivery of antigens by mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive properties. Nevertheless, it has been previously reported that MSCs might also trigger the immune response. We studied whether MSCs may act as carriers, capturing antigens that can be endocytosed by antigen-presenting cells later on. METHODS: We measured the cellular uptake of mannose receptor-mediated fluid phase macropinocytosis, assessed as cellular uptake of fluorescein isothiocyanate-dextran, and PKH-67-labeled cell lysates as a surrogate marker for antigen capture among dendritic cells (DCs, positive control), T lymphocytes (negative control) and MSCs. RESULTS: All experiments confirmed that MCSs displayed pinocytic and endocytic capacities, which were lower than those observed for DCs but significantly higher than those observed for T cells. We also demonstrated that MSCs release previously endocytosed antigens, which subsequently can be captured by DCs. CONCLUSIONS: MSCs have the ability to capture and release antigens.

Better prognosis for patients with del(7q) than for patients with monosomy 7 in myelodysplastic syndrome.

BACKGROUND: Abnormalities involving chromosome 7 are frequent in myelodysplastic syndrome (MDS) and suggest a poor prognosis. METHODS: The authors examined the hypothesis that the clinical features and survival associated with isolated deletion (del) of part of the long arm of chromosome 7 (7q) in MDS are different from those associated with isolated monosomy 7 (complete loss of chromosome 7). In total, 133 patients with a diagnosis of de novo MDS (according to the World Health Organization [WHO] classification) and chromosome 7 abnormalities in the Spanish MDS Registry were evaluated retrospectively. Four karyotypic groups were identified: isolated del(7q) (n = 29), isolated monosomy 7 (n = 27), del(7q) with additional abnormalities (n = 24), and monosomy 7 with additional abnormalities (n = 53). RESULTS: Isolated del(7q) was more frequent in patients with less advanced MDS according to the WHO classification or the International Prognostic Scoring System. In addition, isolated del(7q) was associated with fewer blasts in bone marrow than other cytogenetics groups. Survival was significantly superior in patients with isolated del(7) than in those with isolated monosomy 7, del(7q) with additional abnormalities, or monosomy 7 with additional abnormalities. On multivariate analysis, age, the percentage of blasts in bone marrow, and other chromosome 7 abnormalities apart from isolated del(7q) were identified as independent risk factors for survival. CONCLUSIONS: The current results demonstrated that patients who had MDS with isolated del(7q) had some distinct clinical-pathologic characteristics as well as better survival than patients who had MDS with isolated monosomy 7.

Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting.

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G(0) to G(1) phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.

The novel combination of sirolimus and bortezomib prevents graft-versus-host disease but maintains the graft-versus-leukemia effect after allogeneic transplantation.

BACKGROUND: We have previously shown that bortezomib induces a depletion of alloreactive T cells and allows the expansion of T cells with suppressive properties. In the current study, we analyzed the potential synergistic effect of bortezomib in conjunction with sirolimus in order to reduce-graft-versus-host disease without hampering graft-versus-leukemia effect in the allogeneic transplant setting. DESIGN AND METHODS: We evaluated the effect of sirolimus, bortezomib or the combination of both in the proliferation and activation of in vitro stimulated T lymphocytes. Pathways involved in this synergy were also analyzed using Western blot assays. Finally, BALB/c mice receiving C57BL/6 allogeneic donor bone marrow with splenocytes were used to measure in vivo the effect of this novel combination on the risk of graft-versus-host disease. RESULTS: The combination of both drugs synergistically inhibited both activation and proliferation of stimulated T cells. Also, the production of Th1 cytokines (IFN γ, IL-2 and TNF) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of graft-versus-host disease without hampering graft-versus-leukemia effect, as shown in mice receiving graft-versus-host disease prophylaxis with sirolimus plus bortezomib being infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. CONCLUSIONS: The current study reveals a synergistic effect of the combination sirolimus and bortezomib to prevent graft-versus-host disease while maintaining the graft-versus-leukemia effect.

Simultaneous analysis of the expression of 14 genes with individual prognostic value in myelodysplastic syndrome patients at diagnosis: WT1 detection in peripheral blood adversely affects survival.

Several studies have evaluated the prognostic value of the individual expression of certain genes in patients with myelodysplastic syndromes (MDS). However, none of them includes their simultaneous analysis by quantitative polymerase chain reaction (PCR). We evaluated relative expression levels of 14 molecular markers in 193 peripheral blood samples from untreated MDS patients using real-time PCR. Detectable WT1 expression levels, low TET2, and low IER3 gene expression were the only markers showing in univariate analysis a poor prognostic value for all treatment-free (TFS), progression-free (PFS), and overall survival (OS). In multivariate analysis, molecular parameters associated with a shorter TFS were: WT1 detection (p = 0.014), low TET2 (p = 0.002), and low IER3 expression (p = 0.025). WT1 detection (p = 0.006) and low TET2 (p = 0.006) expression were associated with a shorter PFS when multivariate analysis was carried out by including only molecular markers. Molecular values with an independent value in OS were: WT1 detection (p = 0.003), high EVI1 expression (p = 0.001), and undetectatable p15-CDKN2B (p = 0.037). WT1 expressers were associated with adverse clinical-biological features, high IPSS and WPSS scoring, and unfavorable molecular expression profile. In summary, detectable WT1 expression levels, and low TET2 and low IER3 expression in peripheral blood showed a strong association with adverse prognosis in MDS patients at diagnosis. However, WT1 was the only molecular marker displaying an independent prognostic value in both OS and TFS.