RESUMO
BACKGROUND: Several commercial and academic autologous chimeric antigen receptor T-cell (CAR-T) products targeting CD19 have been approved in Europe for relapsed/refractory B-cell acute lymphoblastic leukemia, high-grade B-cell lymphoma and mantle cell lymphoma. Products for other diseases such as multiple myeloma and follicular lymphoma are likely to be approved by the European Medicines Agency in the near future. DESIGN: The European Society for Blood and Marrow Transplantation (EBMT)-Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association collaborated to draft best practice recommendations based on the current literature to support health care professionals in delivering consistent, high-quality care in this rapidly moving field. RESULTS: Thirty-six CAR-T experts (medical, nursing, pharmacy/laboratory) assembled to draft recommendations to cover all aspects of CAR-T patient care and supply chain management, from patient selection to long-term follow-up, post-authorisation safety surveillance and regulatory issues. CONCLUSIONS: We provide practical, clinically relevant recommendations on the use of these high-cost, logistically complex therapies for haematologists/oncologists, nurses and other stakeholders including pharmacists and health sector administrators involved in the delivery of CAR-T in the clinic.
Assuntos
Hematologia , Receptores de Antígenos Quiméricos , Acreditação , Adulto , Medula Óssea , Humanos , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos TRESUMO
Failure of second-generation tyrosine kinase inhibitors (2GTKI) is a challenging situation in patients with chronic myeloid leukemia (CML). Asciminib, recently approved by the US Federal Drug Administration, has demonstrated in clinical trials a good efficacy and safety profile after failure of 2GTKI. However, no study has specifically addressed response rates to asciminib in ponatinib pretreated patients (PPT). Here, we present data on responses to asciminib from 52 patients in clinical practice, 20 of them (38%) with prior ponatinib exposure. We analyzed retrospectively responses and toxicities under asciminib and compared results between PPT and non-PPT patients.After a median follow-up of 30 months, 34 patients (65%) switched to asciminib due to intolerance and 18 (35%) due to resistance to prior TKIs. Forty-six patients (88%) had received at least 3 prior TKIs. Regarding responses, complete cytogenetic response was achieved or maintained in 74% and 53% for non-PPT and PPT patients, respectively. Deeper responses such as major molecular response and molecular response 4.5 were achieved in 65% and 19% in non-PPT versus 32% and 11% in PPT, respectively. Two patients (4%) harbored the T315I mutation, both PPT.In terms of toxicities, non-PPT displayed 22% grade 3-4 TEAE versus 20% in PPT. Four patients (20% of PPT) suffered from cross-intolerance with asciminib as they did under ponatinib.Our data supports asciminib as a promising alternative in resistant and intolerant non-PPT patients, as well as in intolerant PPT patients; the resistant PPT subset remains as a challenging group in need of further therapeutic options.
Assuntos
Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Piridazinas , Antineoplásicos/efeitos adversos , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Humanos , Imidazóis , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis , Piridazinas/efeitos adversos , Estudos RetrospectivosRESUMO
PURPOSE: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use. BACKGROUND: MSC are being increasingly used in clinical trials for a number of diseases. Biosafety demonstration in all cases is mandatory. Unfortunately, current standard karyotyping methods fail to obtain enough number of evaluable metaphases. METHODS AND MATERIALS: In the present work, to optimise the yield of metaphases in MSC expanded in vitro, we have tested several conditions by modifying colcemid concentration (we have tested 0.01, 0.05 and 0.1 µg mL(-1) ) and exposure time (during 5, 15 and 24 h). We further applied these optimised conditions to 61 MSC expansions in GMP conditions for clinical use. RESULTS: Our results show that the highest number of metaphases was obtained when MSC were incubated with 0.05 µg mL(-1) of colcemid overnight (15 h), compared to the remaining experimental conditions. In most cases (59/61 cases) enough number of metaphases was obtained. And what is more relevant, only in one case a karyotypic abnormality was found (trisomy of chromosome 10), and cells were subsequently discarded for clinical use. CONCLUSION: We describe here an optimal method to obtain enough number of metaphases for karyotype analysis of in vitro expanded MSCs, what is essential for their clinical use in cell therapy programmes.
Assuntos
Cariotipagem/métodos , Células-Tronco Mesenquimais/citologia , Metáfase , Células Cultivadas , Feminino , Humanos , MasculinoRESUMO
Damage to the stem cell progenitors caused by the chemotherapy received in patients diagnosed with non-Hodgkin's lymphoma (NHL) may be an important factor limiting progenitor cell mobilization. The aim of the present analysis was to evaluate the effect of the chemotherapy on the different progenitor cell subpopulations obtained in the leukapheresis. For this purpose, a combination of immunophenotype and functional assays has been performed in 26 mobilized peripheral blood (PB) samples from NHL patients and 36 healthy donors. The different progenitor subpopulations analyzed by flow cytometry significantly correlated with the corresponding populations assessed by functional assays in both healthy donors and NHL patients (p<0.05, r>0.5). The number of committed CFU-GM was similar in both groups (p=0.246), but we found significant decrease in the number of BFU-E and more immature progenitors in PB from NHL patients as compared to donors (p<0.05). Moreover, the number of total CFU was significantly lower in NHL patients (p=0.007). Accordingly, CD34+ cells (p=0.018) and CD34+ subpopulations was decreased in NHL patients. Nevertheless, CD90 and CD34 intensity was significantly higher within CD34+ cells from NHL patients as compared to donors. However, although numerically reduced non-committed CD34+ cells are more immature in chemotherapy mobilized NHL patients. In summary, our results show that all NHL hematopoietic progenitors, analyzed by both immunophenotypical and functional approaches, are impaired in leukapheresis products.
Assuntos
Antígenos CD34/biossíntese , Células-Tronco Hematopoéticas/imunologia , Leucaférese/métodos , Linfoma não Hodgkin/sangue , Antígenos Thy-1/biossíntese , Antígenos CD34/análise , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Doadores de Sangue , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Imunofenotipagem , Linfoma não Hodgkin/diagnóstico , Linfoma não Hodgkin/terapia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Antígenos Thy-1/análiseRESUMO
While in bone marrow allogeneic transplantation the infusion of high doses of progenitor stem cells has a favourable impact on outcome, due to a faster hematopoietic and immune recovery, in the peripheral blood allo-setting the infusion of a high number of CD34 cells increases the risk of extensive chronic graft vs. host disease (cGVHD). This higher incidence of extensive cGVHD has an adverse impact on outcome due to a higher transplant related mortality, specially among patients receiving T-cell depleted allogeneic transplantation with myeloablative conditioning. By contrast, patients undergoing reduced intensity conditioning regimen may benefit from increasing higher CD34 + cell doses, especially those categorized as high risk according to disease status at transplant. Thus, the source of progenitors cells, type of conditioning and GVHD prophylaxis, among other factors, may influence the effect of the progenitor cell dose on outcome after allogeneic transplant.
Assuntos
Antígenos CD34 , Células-Tronco Hematopoéticas , Transplante de Células-Tronco de Sangue Periférico/métodos , Condicionamento Pré-Transplante/métodos , Contagem de Células , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Transplante Homólogo , Resultado do TratamentoRESUMO
Traditional models of hematopoiesis have been hierarchical in nature. Over the past 10 years, we have developed data indicating that hematopoiesis is regulated in a continuum with deterministic and stochastic components. We have shown that the most primitive stem cells, as represented by lineage negative rhodamine(low) Hoechst(low) murine marrow cells are continuously or intermittently cycling as determined by in vivo BrdU labeling. When marrow stem cells are induced to transit cell cycle by in vitro exposure to cytokines, either IL-3, IL-6, IL-11, and steel factor or thrombopoietin, FLT3 ligand, and steel factor, they progress through cycle in a highly synchronized fashion. We have determined that when the stem cells progress through a cytokine stimulated cell cycle the homing, engraftment, adhesion protein, global gene expression, and hematopoietic differentiation phenotypes all change in a reversible fashion. This has led to the continuum model, in which, with cycle transit, chromatin is continually changing altering open transcription areas and providing a continually changing landscape of transcriptional opportunity. More recently, we have extended the changing differentiation profiles to differentiation into lung cells and found that non-hematopoietic differentiation also shows cycle related reversibly modulation. These observations all together support a continuum model of stem cell regulation in which the phenotype of the marrow stem cells is continually and reversibly changing over time.
Assuntos
Células da Medula Óssea/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Humanos , Fenótipo , Processos EstocásticosRESUMO
The impact of donor age in patients with acute myeloid leukemia and myelodysplastic syndrome who underwent allogeneic hematopoietic stem cell transplant (HSCT) remains unclear. In the current study, we evaluate 179 consecutive patients who received an HSCT, from January 2000 to January 2013, in our Institution. Most of the HSCT (91%) were HLA-matched. Patient and donor median age were 51 years (18-69) and 47 years (12-75) respectively, and 81 donors (45%) were older than 50 years. The median follow-up was 38 months (range 1-138), Kaplan-Meier estimated 3-year overall survival (OS) was 63% and disease free survival (DFS) was 56%. Interestingly, patients who received an HSCT from a donor older age (>50 y) showed a poorer OS (51% vs 73%; p=0.01), as well as a higher TRM (20% vs 8%; p=0.038) and higher relapse rate (28% vs 39%; p=0.03). In a stratified subanalysis, 3-year estimated OS was significantly lower among patients undergoing an HSCT from >50 years sibling donors compared to those receiving an HSCT from <50 years unrelated donor (54% vs 72%; p<0.001). In summary, we can conclude that receiving an HSCT from a donor over 50 years old is associated with poorer outcome in patients diagnosed with MDS and AML, and this information may be incorporated into the complex process of donor selection.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicas/terapia , Doadores não Relacionados/estatística & dados numéricos , Adolescente , Adulto , Fatores Etários , Idoso , Criança , Feminino , Transplante de Células-Tronco Hematopoéticas/mortalidade , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/mortalidade , Análise de Sobrevida , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
Hypomethylating agents are able to prolong the overall survival of some patients diagnosed with acute myeloid leukemia. The aim of this study was to evaluate the clinical use of azacitidine as front-line therapy in unfit acute myeloid leukemia (AML) patients and to develop a clinical prediction model to identify which patients may benefit more from the drug. One hundred and ten untreated unfit AML patients received front-line azacitidine therapy in Spain, and response and survival were evaluated in them following European LeukemiaNet (ELN) guidelines. A clinical prediction rule was obtained from this population that was validated and refined in 261 patients treated in France, Austria and Italy. ELN response was achieved in 21.0% of the 371 patients (CI95% 17.0-25.5) and did not depend on bone marrow blast cell percentage. Median overall survival was 9.6 months (CI95% 8.5-10.8) and 40.6% of the patients were alive at 1 year (CI95% 35.5-45.7). European ALMA score (E-ALMA), based on performance status, white blood cell counts at azacitidine onset and cytogenetics, discriminated three risk groups with different survival and response rates. Azacitidine seems a reasonable therapeutic option for most unfit AML patients, i.e. those displaying a favorable or intermediate E-ALMA score.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do Tratamento , Estudos de Validação como AssuntoRESUMO
Immunophenotyping has become an essential tool for diagnosis of hematological malignancies. By contrast, for diagnosis of Waldenstrom's macroglobulinemia (WM) immunophenotyping is used only occasionally. From 150 patients with a IgM monoclonal gammopathy we have selected 60 cases with (1) morphological lymphoplasmocytoid bone marrow (BM) infiltration (>20%); (2) IgM paraprotein (>10g/L); and (3) absence of features of other lymphoma types. Immunophenotypic analysis was based on the use of the triple or quadruple monoclonal antibody (MoAb) combinations. To increase the sensitivity of the analysis of antigen expression, selected CD19(+)CD20(+) B cells were targeted. We have also explored the antigenic characteristics of both the plasma cell (PC) and mast cell (MC) compartments present in the BM from 15 WM patients. Clonal WM lymphocytes were characterized by the constant expression of pan-B markers (CD19, CD20, CD22, CD24) together with sIg, predominantly kappa (5:1, kappa:lambda ratio). A high proportion of cases (75%) were positive for FMC7 and CD25, but in contrast to hairy cell leukemia (HCL), these lymphocytes were always negative for CD103 and CD11c. CD10 antigen was also absent in all WM patients and less than one fifth of patients were positive for CD5 and CD23, while CD27, CD45RA, and BCL-2 were present in most malignant cells. In two cases, the coexistence of two different clones of B lymphocytes was identified, and in eight additional cases, intraclonal phenotypic heterogeneity was observed. As far as PCs are concerned, in most patients (85%) the number of PCs was within the normal range (median, 0.36%). The antigenic profile of these PCs differed from that observed in normal and myelomatous PC (CD38(++)CD19(++/-)CD56(-)CD45(++)CD20(+)). In three cases, PCs showed aberrant expression for CD5, CD22, or FMC7. Finally, the number of mast cells was significantly higher (0.058 +/- 0.13) as compared to normal BM (0.019 +/- 0.02) (P <.01), although they were immunophenotypically normal (CD117(+)CD2(-)CD25(-)).
Assuntos
Macroglobulinemia de Waldenstrom/imunologia , Macroglobulinemia de Waldenstrom/patologia , Células da Medula Óssea , Humanos , Imunoglobulina M/imunologia , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/patologia , Subpopulações de Linfócitos , Linfócitos , Mastócitos , Fenótipo , PlasmócitosAssuntos
Benzoatos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Hidrazinas/administração & dosagem , Pirazóis/administração & dosagem , Trombocitopenia/tratamento farmacológico , Adulto , Idoso , Aloenxertos , Benzoatos/efeitos adversos , Feminino , Humanos , Hidrazinas/efeitos adversos , Masculino , Pessoa de Meia-Idade , Pirazóis/efeitos adversos , Estudos Retrospectivos , Trombocitopenia/sangue , Trombocitopenia/etiologiaRESUMO
Transplantation-associated thrombotic microangiopathy (TA-TMA) is a feared complication of allogeneic hematopoietic SCT (HSCT) owing to its high mortality rate. The use of calcineurin inhibitors or sirolimus (SIR) for GVHD prophylaxis has been suggested as a potential risk factor. However, the impact of tacrolimus (TAC) and SIR combinations on the increased risk of TA-TMA is currently not well defined. We retrospectively analyzed the incidence of TA-TMA in 102 allogeneic HSCT recipients who consecutively received TAC plus SIR (TAC/SIR) (n=68) or plus MTX (TAC/MTX)±ATG (n=34) for GVHD prophylaxis. No significant differences were observed in the incidence of TA-TMA between patients receiving TAC/SIR vs TAC/MTX±ATG (7.4% vs 8.8%, P=0.8). Only grade III-IV acute GVHD, previous HSCT and serum levels of TAC >25 ng/mL were associated with a greater risk of TA-TMA. Patients developing TA-TMA have significantly poorer survival (P<0.001); however, TA-TMA ceased to be an independent prognostic factor when it was included in a multivariate model. In conclusion, the combination of TAC/SIR does not appear to pose a higher risk of TA-TMA. By contrast, we identified three different risk groups for developing TA-TMA.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Metotrexato/administração & dosagem , Sirolimo/administração & dosagem , Tacrolimo/administração & dosagem , Microangiopatias Trombóticas/etiologia , Adulto , Idoso , Quimioterapia Combinada , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/epidemiologia , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/sangue , Incidência , Masculino , Metotrexato/sangue , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Sirolimo/sangue , Tacrolimo/sangue , Microangiopatias Trombóticas/epidemiologia , Transplante Homólogo , Adulto JovemRESUMO
Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) progenitors is increasingly being used. One of the problems that may arise after UCB transplantation is an impaired engraftment. Either intrabone (IB) injection of hematopoietic progenitors or mesenchymal stem cell (MSC) coadministration has been proposed among the strategies to improve engraftment. In the current study, we have assessed the effects of both approaches. Thus, NOD/SCID recipients were transplanted with human UCB CD34+ cells administered either intravenously (IV) or IB, receiving or not bone marrow (BM)-derived MSCs also IV or IB (in the right femur). Human HSC engraftment was measured 3 and 6 weeks after transplantation. Injected MSCs were tracked weekly by bioluminescence. Also, lodgment within the BM niche was assessed at the latter time point by immuno-fluorescence. Our study shows regarding HSC engraftment that the number of BM human CD45+ cells detected 3 weeks after transplantation was significantly higher in mice cotransplanted with human MSCs. Moreover, these mice had a higher myeloid (CD13+) engraftment and a faster B-cell (CD19+) chimerism. At the late time point evaluated (6 weeks), human engraftment was higher in the group in which both strategies were employed (IB injection of HSC and MSC coadministration). When assessing human MSC administration route, we were able to track MSCs only in the injected femurs, whereas they lost their signal in the contralateral bones. These human MSCs were mainly located around blood vessels in the subendosteal region. In summary, our study shows that MSC coadministration can enhance HSC engraftment in our xenogenic transplantation model, as well as IB administration of the CD34+ cells does. The combination of both strategies seems to be synergistic. Interestingly, MSCs were detected only where they were IB injected contributing to the vascular niche.
Assuntos
Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Adulto , Animais , Antígenos CD34/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Osso e Ossos/fisiologia , Quimerismo , Feminino , Sobrevivência de Enxerto/imunologia , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Antígenos Comuns de Leucócito/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante HeterólogoAssuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunossupressores/uso terapêutico , Condicionamento Pré-Transplante/métodos , Adolescente , Adulto , Idoso , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/mortalidade , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Sirolimo/uso terapêutico , Análise de Sobrevida , Tacrolimo/uso terapêutico , Transplante HomólogoRESUMO
Objetivo: Evaluar la presencia de células mesenquimales en el núcleo pulposo de disco intervertebral cervical y caracterizarlas comparativamente con las obtenidas de médula ósea de los mismos sujetos. Pacientes y metodología: Hemos realizado un estudio descriptivo con 14 pacientes que precisaron cirugía de artrodesis cervical. Se analizó la presencia de células mesenquimales (CSM) en el núcleo pulposo (NP) del disco, comparándolas cualitativamente con las de médula ósea (MO) de los mismos pacientes. Se aislaron y expandieron CSM, tanto de NP como de MO. Se realizaron los estudios de diferenciación multilineal in vitro de las células mesenquimales de ambas fuentes, hacia osteoblasto y adipocito, y caracterización inmunofenotípica por citometría de flujo. Resultados: Las células de ambos orígenes se diferencian in vitro hacia ambos tipos celulares, si bien la diferenciación adipocítica de las células procedentes del disco fue menor que las procedentes de MO. Tampoco se han demostrado diferencias en los marcadores inmunofenotípicos. Las células de ambas fuentes poseen los marcadores inmunofenotípicos característicos de las células mesenquimales. Conclusión: El NP de disco vertebral cervical degenerado contiene células troncales mesenquimales. Estas células son similares a las células de MO, con la excepción de su capacidad disminuida de diferenciación adipogénica (AU)
Objective: To evaluate the presence of mesenchymal cells in the nucleus pulposus (NP) of cervical discs and characterize them in comparison ot those obtained from the bone marrow of the same subjects. Patients and methods: We have performed a descriptive study with 14 patients requiring cervical fusion surgery. The presence of mesenchymal stem cells (MSCs) were analyzed in the NP from the disk and compared qualitatively with the bone marrow (BM) of the same patients. MSC were isolated and expanded for both NP and MO. We performed in vitro differentiation studies of mesenchymal cells from both sources, into osteogenic and adipogenic lines, and flow cytometric immunophenotyping. Results: We got differentiation towards both cell types, although adipocyte differentiation of disc-derived cells was decreased compared to those from BM. There were no differences in immunophenotypic markers. Cells from both sources have immunophenotypic markers characteristic of mesenchymal cells. Conclusion: The NP of degenerated cervical disc contains mesenchymal stem cells. These cells are quite similar to BM cells, with the exception of a diminished adipogenic differentiation capacity (AU)
Assuntos
Humanos , Masculino , Feminino , Células-Tronco , Disco Intervertebral/patologia , Disco Intervertebral/cirurgia , Disco Intervertebral , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos , Degeneração do Disco Intervertebral/complicações , Degeneração do Disco Intervertebral/diagnóstico , Degeneração do Disco Intervertebral/cirurgia , Artrodese/métodos , Artrodese/tendências , Artrodese , Terapia Baseada em Transplante de Células e Tecidos/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/tendências , Degeneração do Disco Intervertebral/fisiopatologia , Degeneração do Disco Intervertebral/reabilitação , Degeneração do Disco IntervertebralRESUMO
It is an open question whether in multiple myeloma (MM) bone marrow stromal cells contain genomic alterations, which may contribute to the pathogenesis of the disease. We conducted an array-based comparative genomic hybridization (array-CGH) analysis to compare the extent of unbalanced genomic alterations in mesenchymal stem cells from 21 myeloma patients (MM-MSCs) and 12 normal donors (ND-MSCs) after in vitro culture expansion. Whereas ND-MSCs were devoid of genomic imbalances, several non-recurrent chromosomal gains and losses (>1 Mb size) were detected in MM-MSCs. Using real-time reverse transcription PCR, we found correlative deregulated expression for five genes encoded in regions for which genomic imbalances were detected using array-CGH. In addition, only MM-MSCs showed a specific pattern of 'hot-spot' regions with discrete (<1 Mb) genomic alterations, some of which were confirmed using fluorescence in situ hybridization (FISH). Within MM-MSC samples, unsupervised cluster analysis did not correlate with particular clinicobiological features of MM patients. We also explored whether cytogenetic abnormalities present in myelomatous plasma cells (PCs) were shared by matching MSCs from the same patients using FISH. All MM-MSCs were cytogenetically normal for the tested genomic alterations. Therefore we cannot support a common progenitor for myeloma PCs and MSCs.
Assuntos
Hibridização Genômica Comparativa , Células-Tronco Mesenquimais/química , Mieloma Múltiplo/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Células da Medula Óssea/química , Linhagem da Célula , Células Cultivadas/química , Análise por Conglomerados , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Células-Tronco Neoplásicas/química , Análise de Sequência com Séries de Oligonucleotídeos , Plasmócitos/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/químicaRESUMO
The presence of cytogenetic aberrations on mesenchymal stem cells (MSC) from myelodysplastic syndrome (MDS) patients is controversial. The aim of the study is to characterize bone marrow (BM) derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridization (FISH) and genetic changes by array-based comparative genomic hybridization (array-CGH). In all 36 cases of untreated MDS were studied. MDS-MSC achieved confluence at a significantly slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic. These results were confirmed by FISH. To confirm that genomic changes were also present in freshly obtained MSCs they were enriched by sorting BM cells with the following phenotype: CD45(-)/CD73(++)/CD34(-)/CD271(++). They also showed genomic changes that were confirmed by FISH. To analyze the relationship of these aberrations with clinical-biological data an unsupervised hierarchical cluster analysis was performed, two clusters were identified: the first one included the 5q- syndrome patients, whereas the other incorporated other MDS. Our results show, for the first time that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to a particular MDS subtype, the 5q- syndrome.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 5 , Células-Tronco Mesenquimais/patologia , Síndromes Mielodisplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/patologia , Exame de Medula Óssea , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genéticaRESUMO
We present a patient with critical limb ischemia who was successfully treated with the injection of autologous peripheral blood (PB) CD133+ purified stem cells (SC) into the gastrocnemius muscle. No serious adverse events related to G-CSF administration, mononuclear cells harvest or CD133+ SC administration was observed. After 17 months of follow-up, our patient has experienced limb salvage, symptomatic relief and functional improvement. Moreover, we have observed the appearance of flow in the right posterior tibial artery that was absent before the procedure. To our knowledge, this is the first case of critical limb ischemia treated with PB CD133+ SC.
Assuntos
Antígenos CD/metabolismo , Células Endoteliais/transplante , Extremidades/irrigação sanguínea , Glicoproteínas/metabolismo , Isquemia/cirurgia , Peptídeos/metabolismo , Transplante de Células-Tronco/métodos , Antígeno AC133 , Idoso , Células Endoteliais/metabolismo , Seguimentos , Humanos , Masculino , Resultado do TratamentoRESUMO
BACKGROUND: The aim of this study was to identify circulating endothelial progenitor cells (EPC) with colony-forming capacity and compare them with the monocytic-macrophage lineage. METHODS: Forty-two healthy donors were analyzed. EPC were cultured with VEGF and b-FGF. Sequential studies were performed on days +7 (colonies) +21 and +35. Monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively by adding IGF. RESULTS: The number of EPC colonies was higher in BM than in mobilized or steady-state PB. Using EPC medium, monocytic cells formed cord-like structures but no colonies. However, colonies grew when IGF was added to the medium. By immunocytochemistry, colonies showed CD45, CD31 and lysozyme but no vWF. Colonies were CD4+, CD13+dim, CD14+, CD15++, CD16-/+dim, CD31+dim, CD33+dim, CD45+, CD105-/+dim, lysozyme+ and VE-cadherin+, and constantly negative for CD34, CD133 and KDR, when flow cytometry was used. The immunophenotype of pre-cultured and cultured monocytes was similar to that described for EPC. DISCUSSION: Our results suggest that the so-called 'EPC' obtained at 7 days of culture belong to the monocyte-macrophage lineage, as they share immunophenotypic and molecular features.