RESUMO
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.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 5 , Lenalidomida , Síndromes Mielodisplásicas , Talidomida , Humanos , Lenalidomida/uso terapêutico , Lenalidomida/efeitos adversos , Lenalidomida/administração & dosagem , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Masculino , Feminino , Idoso , Método Duplo-Cego , Pessoa de Meia-Idade , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Talidomida/efeitos adversos , Talidomida/administração & dosagem , Transfusão de Sangue , Idoso de 80 Anos ou mais , Resultado do TratamentoRESUMO
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.
Assuntos
Antígenos CD34/metabolismo , Células da Medula Óssea/metabolismo , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Humanos , CamundongosRESUMO
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.
Assuntos
Dano ao DNA/efeitos dos fármacos , Deferasirox/uso terapêutico , Quelantes de Ferro/uso terapêutico , Síndromes Mielodisplásicas/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Deferasirox/farmacologia , Humanos , Quelantes de Ferro/farmacologia , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Estudos Prospectivos , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Adulto JovemRESUMO
There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells.
Assuntos
Neoplasias Hematológicas/patologia , Janus Quinase 2/metabolismo , Células-Tronco Mesenquimais/fisiologia , Adulto , Idoso , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Expressão Gênica , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/enzimologia , Hematopoese , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Taxa de Sobrevida , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Masculino , Estudos Prospectivos , Espanha/epidemiologiaRESUMO
BACKGROUND: Particularly since the advent of lenalidomide, lower-risk myelodysplastic syndromes (MDS) patients with del(5q) have been the focus of many studies; however, the impact of age on disease characteristics and response to lenalidomide has not been analyzed. METHODS: We assessed the effect of age on clinical characteristics and outcomes in 286 lenalidomide-treated MDS patients with del(5q) from two multicenter trials. RESULTS: A total of 33.9, 34.3, and 31.8% patients were aged <65 years, ≥65 to <75 years, and ≥75 years, respectively. Age <65 years was associated with less favorable International Prognostic Scoring System (IPSS) risk and additional cytopenias at baseline versus older age groups, significantly lower cytogenetic response rates (p = 0.022 vs. ≥65 to <75 years; p = 0.047 vs. ≥75 years), and higher rates of acute myeloid leukemia (AML) progression (Gray's test, p = 0.013). Lenalidomide was equally well tolerated across age groups, producing consistently high rates of red blood cell transfusion independence ≥26 weeks. CONCLUSIONS: Baseline disease characteristics and AML progression appear to be more severe in younger lower-risk MDS patients with del(5q), whereas older age does not seem to compromise the response to lenalidomide. TRIAL REGISTRATION: ClinicalTrials.gov NCT00065156 and NCT00179621.
Assuntos
Fatores Imunológicos/uso terapêutico , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia , Talidomida/análogos & derivados , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Cromossomos Humanos Par 5/genética , Citogenética , Progressão da Doença , Transfusão de Eritrócitos , Feminino , Humanos , Lenalidomida , Leucemia Mieloide Aguda/etiologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Prognóstico , Fatores de Risco , Talidomida/uso terapêutico , Resultado do TratamentoRESUMO
Histone deacetylases (HDACs) are involved in epigenetic modulation and their aberrant expression has been demonstrated in myeloproliferative neoplasms (MPN). HDAC8 inhibition has been shown to inhibit JAK2/STAT5 signaling in hematopoietic cells from MPN. Nevertheless, the role of HDAC8 expression in bone marrow-mesenchymal stromal cells (BM-MSC) has not been assessed. In the current work we describe that HDAC8 is significantly over-expressed in MSC from in JAK-2 positive MPN compared to those from healthy-donors (HD-MSC). Using a selective HDAC8 inhibitor (PCI34051), we verified that the subsequent decrease in the protein and mRNA expression of HDAC8 is linked with an increased apoptosis of malignant MSC whereas it has no effects on normal MSC. In addition, HDAC8 inhibition in MPN-MSC also decreased their capacity to maintain neoplastic hematopoiesis, by increasing the apoptosis, cell-cycle arrest and colony formation of JAK2+-hematopoietic cells. Mechanistic studies using different MPN cell lines revealed that PCI34051 induced their apoptosis, which is enhanced when were co-cultured with JAK2V617F-MSC, decreased their colony formation and the phosphorylation of STAT3 and STAT5. In summary, we show for the first time that the inhibition of HDAC8 in MSC from JAK2+ MPN patients selectively decreases their hematopoietic-supporting ability, suggesting that HDAC8 may be a potential therapeutic target in this setting by acting not only on hematopoietic cells but also on the malignant microenvironment.
Assuntos
Histona Desacetilases/genética , Janus Quinase 2/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transtornos Mieloproliferativos/genética , Proteínas Repressoras/genética , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Hematopoese/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Janus Quinase 2/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Terapia de Alvo Molecular , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismoRESUMO
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.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Tecido Adiposo/citologia , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Análise por Conglomerados , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Humanos , Células-Tronco Mesenquimais/citologia , Especificidade de Órgãos/genética , Placenta/citologia , GravidezRESUMO
BACKGROUND: Graft-versus-host disease (GvHD) remains the major obstacle to successful allogeneic hematopoietic stem cell transplantation, despite of the immunosuppressive regimens administered to control T cell alloreactivity. PI3K/AKT/mTOR pathway is crucial in T cell activation and function and, therefore, represents an attractive therapeutic target to prevent GvHD development. Recently, numerous PI3K inhibitors have been developed for cancer therapy. However, few studies have explored their immunosuppressive effect. METHODS: The effects of a selective PI3K inhibitor (BKM120) and a dual PI3K/mTOR inhibitor (BEZ235) on human T cell proliferation, expression of activation-related molecules, and phosphorylation of PI3K/AKT/mTOR pathway proteins were analyzed. Besides, the ability of BEZ235 to prevent GvHD development in mice was evaluated. RESULTS: Simultaneous inhibition of PI3K and mTOR was efficient at lower concentrations than PI3K specific targeting. Importantly, BEZ235 prevented naïve T cell activation and induced tolerance of alloreactive T cells, while maintaining an adequate response against cytomegalovirus, more efficiently than BKM120. Finally, BEZ235 treatment significantly improved the survival and decreased the GvHD development in mice. CONCLUSIONS: These results support the use of PI3K inhibitors to control T cell responses and show the potential utility of the dual PI3K/mTOR inhibitor BEZ235 in GvHD prophylaxis.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Ativação Linfocitária/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Proliferação de Células/efeitos dos fármacos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
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.
Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/fisiologia , Imunomodulação/fisiologia , Células-Tronco Mesenquimais/fisiologia , Linfócitos T/imunologia , Adulto , Idoso , Células da Medula Óssea/citologia , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Feminino , Humanos , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Doadores de TecidosRESUMO
PURPOSE: This international phase III, randomized, placebo-controlled, double-blind study assessed the efficacy and safety of lenalidomide in RBC transfusion-dependent patients with International Prognostic Scoring System lower-risk non-del(5q) myelodysplastic syndromes ineligible for or refractory to erythropoiesis-stimulating agents. PATIENTS AND METHODS: In total, 239 patients were randomly assigned (2:1) to treatment with lenalidomide (n = 160) or placebo (n = 79) once per day (on 28-day cycles). The primary end point was the rate of RBC transfusion independence (TI) ≥ 8 weeks. Secondary end points were RBC-TI ≥ 24 weeks, duration of RBC-TI, erythroid response, health-related quality of life (HRQoL), and safety. RESULTS: RBC-TI ≥ 8 weeks was achieved in 26.9% and 2.5% of patients in the lenalidomide and placebo groups, respectively (P < .001). Ninety percent of patients achieving RBC-TI responded within 16 weeks of treatment. Median duration of RBC-TI with lenalidomide was 30.9 weeks (95% CI, 20.7 to 59.1). Transfusion reduction of ≥ 4 units packed RBCs, on the basis of a 112-day assessment, was 21.8% in the lenalidomide group and 0% in the placebo group. Higher response rates were observed in patients with lower baseline endogenous erythropoietin ≤ 500 mU/mL (34.0% v 15.5% for > 500 mU/mL). At week 12, mean changes in HRQoL scores from baseline did not differ significantly between treatment groups, which suggests that lenalidomide did not adversely affect HRQoL. Achievement of RBC-TI ≥ 8 weeks was associated with significant improvements in HRQoL (P < .01). The most common treatment-emergent adverse events were neutropenia and thrombocytopenia. CONCLUSION: Lenalidomide yields sustained RBC-TI in 26.9% of RBC transfusion-dependent patients with lower-risk non-del(5q) myelodysplastic syndromes ineligible for or refractory to erythropoiesis-stimulating agents. Response to lenalidomide was associated with improved HRQoL. Treatment-emergent adverse event data were consistent with the known safety profile of lenalidomide.
Assuntos
Transfusão de Eritrócitos , Síndromes Mielodisplásicas/terapia , Talidomida/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Método Duplo-Cego , Resistência a Medicamentos , Eritropoese/efeitos dos fármacos , Feminino , Expressão Gênica , Humanos , Lenalidomida , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Qualidade de Vida , Talidomida/efeitos adversos , Talidomida/uso terapêuticoRESUMO
The biological and molecular events that underlie bone marrow fibrosis in patients with myelodysplastic syndromes are poorly understood, and its prognostic role in the era of the Revised International Prognostic Scoring System (IPSS-R) is not yet fully determined. We have evaluated the clinical and biological events that underlie bone marrow fibrotic changes, as well as its prognostic role, in a well-characterized prospective patient cohort (n=77) of primary MDS patients. The degree of marrow fibrosis was linked to parameters of erythropoietic failure, marrow cellularity, p53 protein accumulation, WT1 gene expression, and serum levels of CXCL9 and CXCL10, but not to other covariates including the IPSS-R score. The presence of bone marrow fibrosis grade 2 or higher was associated with the presence of mutations in cohesin complex genes (31.5% vs. 5.4%, p=0.006). By contrast, mutations in CALR, JAK2, PDGFRA, PDGFRB,and TP53 were very rare. Survival analysis showed that marrow fibrosis grade 2 or higher was a relevant significant predictor for of overall survival, and independent of age, performance status, and IPSS-R score in multivariate analysis.
Assuntos
Medula Óssea/metabolismo , Análise Mutacional de DNA/métodos , Mutação , Síndromes Mielodisplásicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Quimiocina CXCL10/sangue , Quimiocina CXCL9/sangue , Feminino , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Prognóstico , Estudos Prospectivos , Análise de Sobrevida , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
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.
Assuntos
Tecido Adiposo/citologia , Comunicação Celular/imunologia , Imunomodulação , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adulto , Idoso , Biomarcadores , Ciclo Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Humanos , Imunofenotipagem , Ativação Linfocitária , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , FenótipoRESUMO
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.
Assuntos
5'-Nucleotidase/análise , Vesículas Extracelulares/química , Citometria de Fluxo/métodos , Receptores de Hialuronatos/análise , Células-Tronco Mesenquimais/citologia , Antígenos Thy-1/análise , Adulto , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/química , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: We aimed to compare the ability of recently developed prognostic indices for myelodysplastic syndromes to identify patients with poor prognoses within the lower-risk (low and intermediate-1) categories defined by the International Prognosis Scoring System (IPSS). METHODS: We included patients with de-novo myelodysplastic syndromes diagnosed between Nov 29, 1972, and Dec 15, 2011, who had low or intermediate-1 IPSS scores and were in the Spanish Registry of Myelodysplastic Syndromes. We reclassified these patients with the new prognostic indices (revised IPSS [IPSS-R], revised WHO-based Prognostic Scoring System [WPSS-R], Lower Risk Scoring System [LRSS], and the Grupo Español de Síndromes Mielodisplásicos [Spanish Group of Myelodysplastic Syndromes; GESMD]) and calculated the overall survival of the different risk groups within each prognostic index to identify the groups of patients with overall poor prognoses (defined as an expected overall survival <30 months). We calculated overall survival with the Kaplan-Meier method. FINDINGS: We identified 2373 patients. None of the prognostic indices could be used to identify a population with poor prognoses (median overall survival <30 months) for the patients with low IPSS scores (1290 individuals). In the group with intermediate-1 scores (1083 individuals), between 17% and 47% of patients were identified as having poor prognoses with the new prognostic indices. The LRSS had the best model fit with the lowest value in the Akaike information criteria test, whereas the IPSS-R identified the largest proportion of patients with poor prognoses (47%). Patients with intermediate-1 scores who were classified as having poor prognoses by one or more prognostic index (646 [60%] individuals) had worse median overall survival (33·1 months, 95% CI 28·4-37·9) than did patients who were classified as having low risk by all prognostic indices (63·7 months, 49·5-78·0], HR 1·9, 95% CI 1·6-2·3, p<0·0001) INTERPRETATION: Recently proposed prognostic indices for myelodysplastic syndromes can be used to improve identification of patients with poor prognoses in the group of patients with intermediate-1 IPSS scores, who could potentially benefit from a high-risk treatment approach. FUNDING: None.
Assuntos
Síndromes Mielodisplásicas/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/classificação , Prognóstico , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Análise de SobrevidaRESUMO
Sixty-seven-year-old patient, diagnosed with multiple myeloma who had received autologous stem cell transplantation, following bortezomib, dexamethasone and thalidomide conventional regimen, achieving complete response, developed rash, diarrhea, and severe respiratory failure, 80 days after the transplantation procedure. He was diagnosed with graft-versus-host disease and bronchiolitis obliterans syndrome.
RESUMO
The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/patologia , Vesículas Secretórias/fisiologia , Células Cultivadas , Células Clonais/metabolismo , Células Clonais/patologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismoRESUMO
Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP(+)-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP(-) cells, which confirms their differentiation into corneal tissues. GFP(+)-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD.
Assuntos
Células da Medula Óssea/citologia , Córnea/citologia , Transplante de Córnea/efeitos adversos , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Adulto , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Proteínas da Matriz Extracelular/metabolismo , Feminino , Doença Enxerto-Hospedeiro/terapia , Proteínas de Fluorescência Verde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Placenta/citologia , Adulto , Antígenos CD/genética , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Separação Celular , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Pessoa de Meia-Idade , Gravidez , Fatores de Transcrição/metabolismoRESUMO
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).