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Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.
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Linfoma de Células B , Macroglobulinemia de Waldenström , Humanos , Animales , Ratones , Macroglobulinemia de Waldenström/patología , Ligando de CD40/genética , Fosfatidilinositol 3-Quinasas , Ligandos , Transducción de Señal , Linfoma de Células B/complicaciones , Microambiente TumoralRESUMEN
Therapy-related myeloid neoplasm (t-MN) is a threatening complication of autologous stem cell transplantation (ASCT). Detecting clonal hematopoiesis (CH) mutations in cryopreserved cells before ASCT has been associated with a higher risk of t-MN, but the evolution of molecular abnormalities from pre-ASCT to t-MN, within the same patient, remains to be elucidated. We evaluated the mutational profile of 19 lymphoma/myeloma patients, at both pre-ASCT and t-MN diagnosis, using a targeted NGS approach; 26 non-developing t-MN control patients were also studied pre-ASCT. At ASCT, we found a higher frequency of CH in patients developing t-MN (58%) than in those who did not (23%) (P = 0.029); mutations in epigenetic (DNMT3A, TET2, and ASXL1) and DNA repair genes (PPM1D, RAD21, TP53, and STAG2) were the most represented. At t-MN, CH increased to 82% of patients. Cumulative mutational burden and variant allele frequency (VAF) also increased at t-MN. CH clones detected at ASCT were found at t-MN in eight out of 16 patients, mainly with stable VAF. Among the new driver mutations appeared at t-MN, TP53 increased from one to 13 mutations, in nine patients; being associated with complex karyotype. Mutations in transcription factor (RUNX1, CEBPA) and intracellular signaling genes (FLT3, RAS genes) also increased from three to 17 mutations in eight patients, presenting with a normal karyotype. Overall, we found that preexisting CH at ASCT rarely causes t-MN directly, but may rather facilitate the appearance of new mutations, especially those involving TP53, RUNX1, and RAS, that can drive the evolution to t-MN of at least two distinct types.
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Trasplante de Células Madre Hematopoyéticas , Trastornos Mieloproliferativos , Neoplasias Primarias Secundarias , Hematopoyesis Clonal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Mutación , Trastornos Mieloproliferativos/complicaciones , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/terapia , Neoplasias Primarias Secundarias/genética , Trasplante Autólogo/efectos adversosRESUMEN
Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.
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Antígeno AC133/genética , Células Progenitoras Endoteliales/citología , Sangre Fetal/citología , Citometría de Flujo/normas , Inmunofenotipificación/normas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Antígeno AC133/inmunología , Adolescente , Adulto , Anciano , Antígenos CD34/genética , Antígenos CD34/inmunología , Benchmarking , Antígeno CD146/genética , Antígeno CD146/inmunología , Recuento de Células , Células Progenitoras Endoteliales/inmunología , Femenino , Sangre Fetal/inmunología , Colorantes Fluorescentes/química , Expresión Génica , Voluntarios Sanos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Masculino , Persona de Mediana Edad , Guías de Práctica Clínica como Asunto , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Autologous stem cell transplantation (ASCT) is a potentially curative treatment of lymphoma, but peripheral blood stem cell (PBSC) mobilization fails in some patients. PBSC mobilizing agents have recently been proved to improve the PBSC yield after a prior mobilization failure. Predictive parameters of mobilization failure allowing for a preemptive, more cost-effective use of such agents during the first mobilization attempt are still poorly defined, particularly during mobilization with chemotherapy + granulocyte colony-stimulating factor (G-CSF). We performed a retrospective analysis of a series of lymphoma patients who were candidates for ASCT, to identify factors influencing PBSC mobilization outcome. Premobilization parameters-age, histology, disease status, mobilizing protocol, and previous treatments-as well as white blood cell (WBC) and PBSC kinetics, markers potentially able to predict failure during the ongoing mobilization attempt, were analyzed in 415 consecutive mobilization procedures in 388 patients. We used chemotherapy + G-CSF in 411 (99%) of mobilization attempts and PBSC collection failed (<2 × 10(6) CD34+ PBSC/kg) in 13%. Multivariable analysis showed that only a low CD34+ PBSC count and CD34+ PBSC/WBC ratio, together with the use of nonplatinum-containing chemotherapy, independently predicted mobilization failure. Using these three parameters, we established a scoring system to predict risk of failure during mobilization ranging from 2 to 90%, thus allowing a selective use of a preemptive mobilization policy.
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Antineoplásicos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Leucocitos/citología , Linfoma/sangre , Linfoma/terapia , Células Madre/citología , Adolescente , Adulto , Anciano , Antígenos CD34/metabolismo , Femenino , Filgrastim/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estudios Retrospectivos , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND: We aimed to assess the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data. METHODS: We contacted 51 European centres and asked them to provide reported and unreported anonymised data for individual patients with metastatic breast cancer who participated in studies between January, 2003, and July, 2012. Eligible studies had participants starting a new line of therapy, data for progression-free survival or overall survival, or both, and CTC quantification by the CellSearch method at baseline (before start of new treatment). We used Cox regression models, stratified by study, to establish the association between CTC count and progression-free survival and overall survival. We used the landmark method to assess the prognostic value of CTC and serum marker changes during treatment. We assessed the added value of CTCs or serum markers to prognostic clinicopathological models in a resampling procedure using likelihood ratio (LR) χ(2) statistics. FINDINGS: 17 centres provided data for 1944 eligible patients from 20 studies. 911 patients (46·9%) had a CTC count of 5 per 7·5 mL or higher at baseline, which was associated with decreased progression-free survival (hazard ratio [HR] 1·92, 95% CI 1·73-2·14, p<0·0001) and overall survival (HR 2·78, 95% CI 2·42-3·19, p<0·0001) compared with patients with a CTC count of less than 5 per 7·5 mL at baseline. Increased CTC counts 3-5 weeks after start of treatment, adjusted for CTC count at baseline, were associated with shortened progression-free survival (HR 1·85, 95% CI 1·48-2·32, p<0·0001) and overall survival (HR 2·26, 95% CI 1·68-3·03) as were increased CTC counts after 6-8 weeks (progression-free survival HR 2·20, 95% CI 1·66-2·90, p<0·0001; overall survival HR 2·91, 95% CI 2·01-4·23, p<0·0001). Survival prediction was significantly improved by addition of baseline CTC count to the clinicopathological models (progression-free survival LR 38·4, 95% CI 21·9-60·3, p<0·0001; overall survival LR 64·9, 95% CI 41·3-93·4, p<0·0001). This model was further improved by addition of CTC change at 3-5 weeks (progression-free survival LR 8·2, 95% CI 0·78-20·4, p=0·004; overall survival LR 11·5, 95% CI 2·6-25·1, p=0·0007) and at 6-8 weeks (progression-free survival LR 15·3, 95% CI 5·2-28·3; overall survival LR 14·6, 95% CI 4·0-30·6; both p<0·0001). Carcinoembryonic antigen and cancer antigen 15-3 concentrations at baseline and during therapy did not add significant information to the best baseline model. INTERPRETATION: These data confirm the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not. FUNDING: Janssen Diagnostics, the Nuovo-Soldati foundation for cancer research.
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Neoplasias de la Mama/secundario , Células Neoplásicas Circulantes/patología , Anciano , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/terapia , Antígeno Carcinoembrionario/sangre , Recuento de Células , Distribución de Chi-Cuadrado , Supervivencia sin Enfermedad , Europa (Continente) , Femenino , Humanos , Estimación de Kaplan-Meier , Funciones de Verosimilitud , Persona de Mediana Edad , Mucina-1/sangre , Células Neoplásicas Circulantes/metabolismo , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Reproducibilidad de los Resultados , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Human mesenchymal stromal cells (hMSCs), whether used alone or together with three-dimensional scaffolds, are the best-studied postnatal stem cells in regenerative medicine. In this study, innovative composite scaffolds consisting of a core-shell architecture were seeded with bone-marrow-derived hMSCs (BM-hMSCs) and tested for their biocompatibility and remarkable capacity to promote and support bone regeneration and mineralization. The scaffolds were prepared by grafting three different amounts of gelatin-chitosan (CH) hydrogel into a 3D-printed polylactic acid (PLA) core (PLA-CH), and the mechanical and degradation properties were analyzed. The BM-hMSCs were cultured in the scaffolds with the presence of growth medium (GM) or osteogenic medium (OM) with differentiation stimuli in combination with fetal bovine serum (FBS) or human platelet lysate (hPL). The primary objective was to determine the viability, proliferation, morphology, and spreading capacity of BM-hMSCs within the scaffolds, thereby confirming their biocompatibility. Secondly, the BM-hMSCs were shown to differentiate into osteoblasts and to facilitate scaffold mineralization. This was evinced by a positive Von Kossa result, the modulation of differentiation markers (osteocalcin and osteopontin), an expression of a marker of extracellular matrix remodeling (bone morphogenetic protein-2), and collagen I. The results of the energy-dispersive X-ray analysis (EDS) clearly demonstrate the presence of calcium and phosphorus in the samples that were incubated in OM, in the presence of FBS and hPL, but not in GM. The chemical distribution maps of calcium and phosphorus indicate that these elements are co-localized in the same areas of the sections, demonstrating the formation of hydroxyapatite. In conclusion, our findings show that the combination of BM-hMSCs and PLA-CH, regardless of the amount of hydrogel content, in the presence of differentiation stimuli, can provide a construct with enhanced osteogenicity for clinically relevant bone regeneration.
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Induced Pluripotent Stem Cells (iPSCs) are nowadays a common starting point for wide-ranging applications including 3D disease modeling (i.e. organoids) and in future regenerative medicine. Physiological processes like homeostasis, cell differentiation, development and reproduction are tightly regulated by hormones through binding to their transmembrane or nuclear receptors of target cells. Considering their pleiotropic effect, take into account also their expression in an iPSCs-based disease modeling would better recapitulate the molecular events leading to 3D organoid development and disease study. Here we reported the expression pattern of estrogen receptor (ERα) and progesterone receptor (PR) in four different iPSCs, obtained from CD34 + progenitor cells and skin fibroblasts with four different methods. Expression of ERα and PR mRNA were significantly downregulated in iPSCs as well as fibroblasts compared to MCF7 positive control. Immunofluorescence (IF) staining detected only the expression of PR protein in all the different iPSCs cell lines, while ERα was not detectable. By flow cytometry analysis we observed that the ~ 65% of the total population of iPSCs cells expressed only PR, with 100% fold increase compared to HSPCs and fibroblasts, while ERα was not expressed. Our results collectively demonstrated for the first time that the reprogramming of somatic cells into iPSCs leads to the expression of PR receptor.
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Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a rare but potentially fatal complication following allogenic hematopoietic cell transplantation (allo-HCT). Timely identification of SOS/VOD to allow for prompt treatment is critical, but identifying a VOD-predictive biomarker remains challenging. Given the pivotal role of endothelial dysfunction in SOS/VOD pathophysiology, the CECinVOD study prospectively evaluated levels of circulating endothelial cells (CECs) in patients undergoing allo-HCT with a myeloablative conditioning (MAC) regimen to investigate the potential of CEC level in predicting and diagnosing SOS/VOD. A total of 150 patients from 11 Italian bone marrow transplantation units were enrolled. All participants were age >18 years and received a MAC regimen, putting them at elevated risk of developing SOS/VOD. Overall, 6 cases of SOS/VOD (4%) were recorded. CECs were detected using the Food and Drug Administration-approved CellSearch system, an immunomagnetic selection-based platform incorporating ferrofluid nanoparticles and fluorescent-labeled antibodies, and were defined as CD146+, CD105+, DAPI+, or CD45-. Blood samples were collected at the following time points: before (T0) and at the end of conditioning treatment (T1), at neutrophil engraftment (T2), and at 7 to 10 days postengraftment (T3). For patients who developed VOD, additional samples were collected at any suspected or proven VOD onset (T4) and weekly during defibrotide treatment (T5 to T8). A baseline CEC count >17/mL was associated with an elevated risk of SOS/VOD (Pâ¯=â¯.04), along with bilirubin level >1.5 mg/mL and a haploidentical donor hematopoietic stem cell source. Postconditioning regimen (T1) CEC levels were elevated (Pâ¯=â¯.02), and levels were further increased at engraftment (P < .0001). Additionally, patients developing SOS/VOD after engraftment, especially those with late-onset SOS/VOD, showed a markedly higher relative increase (>150%) in CEC count. Multivariate analysis supported these findings, along with a high Endothelial Activation and Stress Index (EASIX) score at engraftment (T2). Finally, CEC kinetics corresponded with defibrotide treatment. After the start of therapy (T4), CEC levels showed an initial increase in the first week (T5), followed by a progressive decrease during VOD treatment (T6 and T7) and a return to pre-SOS/VOD onset levels at resolution of the complication. This prospective multicenter study reveals a low incidence of SOS/VOD in high-risk patients compared to historical data, in line with recent reports. The results from the CECinVOD study collectively confirm the endothelial injury in allo-HCT and its role in in the development of SOS/VOD, suggesting that CEC level can be a valuable biomarker for diagnosing SOS/VOD and identifying patients at greater risk of this complication, especially late-onset SOS/VOD. Furthermore, CEC kinetics may support treatment strategies by providing insight into the optimal timing for discontinuing defibrotide treatment.
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Biomarcadores , Células Endoteliales , Trasplante de Células Madre Hematopoyéticas , Enfermedad Veno-Oclusiva Hepática , Humanos , Enfermedad Veno-Oclusiva Hepática/etiología , Enfermedad Veno-Oclusiva Hepática/sangre , Femenino , Masculino , Células Endoteliales/patología , Células Endoteliales/metabolismo , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Persona de Mediana Edad , Adulto , Biomarcadores/sangre , Acondicionamiento Pretrasplante/efectos adversos , Estudios Prospectivos , Trasplante Homólogo/efectos adversos , Anciano , Polidesoxirribonucleótidos/uso terapéutico , Factores de Riesgo , Adulto JovenRESUMEN
High-dose chemotherapy with autologous peripheral blood stem cell rescue has been reported as feasible and effective in HIV-associated lymphoma. Although a sufficient number of stem cells seems achievable in most patients, there are cases of stem cell harvest failure. The aim of this study was to describe the mobilization policies used in HIV-associated lymphoma, evaluate the failure rate and identify factors influencing mobilization results. We analyzed 155 patients who underwent attempted stem cell mobilization at 10 European centers from 2000-2012. One hundred and twenty patients had non-Hodgkin lymphoma and 35 Hodgkin lymphoma; 31% had complete remission, 57% chemosensitive disease, 10% refractory disease, 2% untested relapse. Patients were mobilized with chemotherapy + G-CSF (86%) or G-CSF alone (14%); 73% of patients collected >2 and 48% >5 × 10(6) CD34(+) cells/kg. Low CD4+ count and refractory disease were associated with mobilization failure. Low CD4(+) count, low platelet count and mobilization with G-CSF correlated with lower probability to achieve >5 × 10(6) CD34(+) cells/kg, whereas cyclophosphamide ≥ 3 g/m(2) + G-CSF predicted higher collections. Circulating CD34(+) cells and CD34/WBC ratio were strongly associated with collection result. HIV infection alone should not preclude an attempt to obtain stem cells in candidates for autologous transplant as the results are comparable to the HIV-negative population.
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Seropositividad para VIH/terapia , Movilización de Célula Madre Hematopoyética/métodos , Enfermedad de Hodgkin/terapia , Linfoma no Hodgkin/terapia , Adulto , Anciano , Femenino , Seropositividad para VIH/diagnóstico , Seropositividad para VIH/epidemiología , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/epidemiología , Humanos , Linfoma no Hodgkin/diagnóstico , Linfoma no Hodgkin/epidemiología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
PURPOSE: Here, we present the procedure to obtain allogeneic solid platelet-rich plasma (PRP) and its use in a pilot study of patients with persistent neurotrophic epithelial defects. METHODS: We included 4 eyes of 4 patients with persistent neurotrophic epithelial defects unresponsive to other therapies from a single institution. PRP and thrombin were produced by the Department of Transfusion Medicine from healthy blood donors. PRP was activated in its solid form in the operating room with addition of thrombin and calcium gluconate 10% and applied on the cornea with fibrin glue and soft contact lens. Corneal healing time, corneal esthesiometry, visual acuity, Oxford staining score, Ocular Surface Disease Index questionnaire, and Schirmer I test were recorded. Anterior segment optical coherence tomography and in vivo confocal microscopy were also evaluated over the 4-month follow-up period. RESULTS: The persistent epithelial defect healed in all patients in the first 10 days. During the follow-up, there was an absence of recurrences. For all patients, there was a reduction in Ocular Surface Disease Index questionnaire score (case 1: -55 points, -73.3%; case 2: -26.3 points, -58.4%; case 3: -56 points, -69.1%; case 4: -20 points, -26.6%; mean reduction: 39.3 points, 56.85%) and Oxford staining score (case 1, 2, and 3: 3 points decrease; case 4: 2 points decrease; mean reduction: -2.75 points). CONCLUSIONS: Allogeneic solid PRP in combination with fibrin glue may facilitate wound healing in neurotrophic persistent epithelial defects. Further prospective studies are needed to quantify its efficacy.
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Trasplante de Células Madre Hematopoyéticas , Plasma Rico en Plaquetas , Humanos , Proyectos Piloto , Adhesivo de Tejido de Fibrina , TrombinaRESUMEN
Background: Chimeric antigen receptor (CAR) T-cell therapy represents the most advanced immunotherapy against relapsed/refractory B cell malignancies. While cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome are distinctive, known CAR T-cell acute adverse events, hematological toxicity has been increasingly reported. Cytopenia following CAR T-cell treatment is attributed in most cases to lymphodepletion regimens, bridging chemotherapy, or radiotherapy. However, when cytopenia becomes prolonged, the development of myelodysplastic syndrome (MDS) should be considered. Case presentation: We report a case of high risk (HR)-MDS following CAR T-cell therapy in a patient with relapsed diffuse large B cell lymphoma. Eight months after CAR T-cell infusion, the blood count showed progressive, worsening cytopenia and the bone marrow biopsy revealed multilineage dysplasia without excess of blasts associated with chromosome 7 deletion and RUNX1 mutation. Next generation sequencing analysis, retrospectively performed on stored samples, showed a germ line CSF3R mutation, CEBPA clonal hematopoiesis, but no RUNX1 lesion. Conclusion: We describe a case of HR-MDS, with deletion of chromosome 7 and acquisition of RUNX1 mutation, developing after CAR T-cell therapy in a patient with clonal hematopoiesis (CH). Previous chemotherapy favored MDS onset; however, we could not exclude the fact that the impairment of immunosurveillance related to either lymphodepletion or CAR T-cell infusion may play a role in MDS development. Thus, we designed a multicenter prospective study (ClonHema-CAR-T-Study) to investigate if cytopenia after CAR T-cell treatment may be due to underling CH as well as the presence of secondary myeloid malignancies.
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Background: The COVID-19 pandemic has had a significant impact on the management and care of onco-hematological patients, particularly those with lymphoproliferative disorders who are at higher risk for COVID-19 associated bacterial and fungal superinfections. Case presentation: We present the successful treatment of a 44-year-old male patient with refractory mantle cell lymphoma treated with chimeric antigen receptor T (CAR-T) cell therapy, despite concurrent COVID-19 infection. The patient developed grade II cytokine release syndrome, requiring admission to the intensive care unit. The CAR-T cells expanded effectively, and the patient achieved complete metabolic remission. During the treatment course, the patient experienced complications including COVID-19-associated pulmonary aspergillosis and a co-infection with Stenotrophomonas maltophilia and the SARS-CoV-2 omicron variant. Prompt antifungal and antibacterial therapy, along with appropriate COVID-19 treatment, led to the resolution of these infections. Dexamethasone was also administered to reduce inflammation and aid hematologic recovery. Despite the presence of multiple infections, the patient achieved complete remission of lymphoma, highlighting the effectiveness of CAR-T cell therapy in this high-risk patient. Conclusion: Despite the challenges posed by concurrent infections, the decision to proceed with CAR-T cell therapy in this patient proved to be successful, resulting in complete remission of lymphoma. Early initiation of supportive therapies and the use of dexamethasone contributed to the resolution of complications. This case underscores the importance of individualized decision-making and the potential benefits of CAR-T cell therapy in similar high-risk patients.
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The development of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematological diseases. However, approximately 60% of patients relapse after CAR-T cell therapy, and no clear cause for this failure has been identified. The objective of the Bio-CAR-T BS study (ClinicalTrials.gov: NCT05366569) is to improve our understanding of the lymphocyte harvest to maximize the quality of the CAR-T cell product. Of the 14 patients enrolled, 11 were diagnosed with DLBCL, 2 with PMBCL, and 1 with ALL. Five of 11 DLBCL patients met the criteria for "pre-emptive" Lymphocytes-apheresis (being at high risk of second relapse), and 6 were included in the standard-of-care Lymphocytes-apheresis group. Previous autologous stem cell transplantation (ASCT) and age were significantly different between the two groups. At the time of Lymphocyte-apheresis, patients in the "pre-emptive" group had more "fit" lymphocytes (higher CD4+/CD8+ ratio; higher naïve T cells levels) compared with standard group, probably due to the impact of ASCT. At the same time, also being older than 60 years results in a more "exhausted" lymphocyte profile. Overall, "pre-emptive" Ly-apheresis in DLBCL patients at high risk of relapse appears to be feasible and may allow the timely collection of "fit" lymphocytes for CAR-T cell manufacturing.
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Human platelet lysate (hPL) is considered a valid substitute to fetal bovine serum (FBS) in the expansion of mesenchymal stromal cells (MSC), and it is commonly produced starting from intermediate side products of whole blood donations. Through freeze-thaw cycles, hPL is highly enriched in chemokines, growth factors, and adhesion and immunologic molecules. Cell therapy protocols, using hPL instead of FBS for the expansion of cells, are approved by regulatory authorities without concerns, and its administration in patients is considered safe. However, published data are fairly difficult to compare, since the production of hPL is highly variable. This study proposes to optimize and standardize the hPL productive process by using instruments, technologies, and quality/safety standards required for blood bank activities and products. The quality and improved selection of the starting material (i.e., the whole blood), together with the improvement of the production process, guarantee a product characterized by higher content and quality of growth factors as well as a reduction in batch-to-batch variability. By increasing the number of freeze/thaw cycles from one (hPL1c) to four (hPL4c), we obtained a favorable effect on the release of growth factors from platelet α granules. Those changes have directly translated into biological effects leading to a decreasing doubling time (DT) of MSC expansion at 7 days (49.41 ± 2.62 vs. 40.61 ± 1.11 h, p < 0.001). Furthermore, mass spectrometry (MS)-based evaluation has shown that the proliferative effects of hPL4c are also combined with a lower batch-to-batch variability (10-15 vs. 21-31%) at the proteomic level. In conclusion, we have considered lot-to-lot hPL variability, and by the strict application of blood bank standards, we have obtained a standardized, reproducible, safe, cheap, and ready-to-use product.
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Gelatin-dextran hydrogel scaffolds (G-PEG-Dx) were evaluated for their ability to activate the bone marrow human mesenchymal stromal cells (BM-hMSCs) towards mineralization. G-PEG-Dx1 and G-PEG-Dx2, with identical composition but different architecture, were seeded with BM-hMSCs in presence of fetal bovine serum or human platelet lysate (hPL) with or without osteogenic medium. G-PEG-Dx1, characterized by a lower degree of crosslinking and larger pores, was able to induce a better cell colonization than G-PEG-Dx2. At day 28, G-PEG-Dx2, with hPL and osteogenic factors, was more efficient than G-PEG-Dx1 in inducing mineralization. Scanning electron microscopy (SEM) and Raman spectroscopy showed that extracellular matrix produced by BM-hMSCs and calcium-positive mineralization were present along the backbone of the G-PEG-Dx2, even though it was colonized to a lesser degree by hMSCs than G-PEG-Dx1. These findings were confirmed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), detecting distinct lipidomic signatures that were associated with the different degree of scaffold mineralization. Our data show that the architecture and morphology of G-PEG-Dx2 is determinant and better than that of G-PEG-Dx1 in promoting a faster mineralization, suggesting a more favorable and active role for improving bone repair.
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Selection of feasible hybrid-hydrogels for best chondrogenic differentiation of human mesenchymal stromal cells (hMSCs) represents an important challenge in cartilage regeneration. In this study, three-dimensional hybrid hydrogels obtained by chemical crosslinking of poly (ethylene glycol) diglycidyl ether (PEGDGE), gelatin (G) without or with chitosan (Ch) or dextran (Dx) polysaccharides were developed. The hydrogels, namely G-PEG, G-PEG-Ch and G-PEG-Dx, were prepared with an innovative, versatile and cell-friendly technique that involves two preparation steps specifically chosen to increase the degree of crosslinking and the physical-mechanical stability of the product: a first homogeneous phase reaction followed by directional freezing, freeze-drying and post-curing. Chondrogenic differentiation of human bone marrow mesenchymal stromal cells (hBM-MSC) was tested on these hydrogels to ascertain whether the presence of different polysaccharides could favor the formation of the native cartilage structure. We demonstrated that the hydrogels exhibited an open pore porous morphology with high interconnectivity and the incorporation of Ch and Dx into the G-PEG common backbone determined a slightly reduced stiffness compared to that of G-PEG hydrogels. We demonstrated that G-PEG-Dx showed a significant increase of its anisotropic characteristic and G-PEG-Ch exhibited higher and faster stress relaxation behavior than the other hydrogels. These characteristics were associated to absence of chondrogenic differentiation on G-PEG-Dx scaffold and good chondrogenic differentiation on G-PEG and G-PEG-Ch. Furthermore, G-PEG-Ch induced the minor collagen proteins and the formation of collagen fibrils with a diameter like native cartilage. This study demonstrated that both anisotropic and stress relaxation characteristics of the hybrid hydrogels were important features directly influencing the chondrogenic differentiation potentiality of hBM-MSC.
Asunto(s)
Células Madre Mesenquimatosas , Diferenciación Celular , Condrogénesis , Gelatina , Humanos , Hidrogeles , Ingeniería de TejidosRESUMEN
A role of endothelial cells (ECs) in Primary Myelofibrosis (PMF) was supposed since JAK2 mutation was found in endothelial precursor cells (EPCs) and in ECs captured by laser microdissection. By Cell Search method, the circulating endothelial cells (CECs) from 14 PMF patients and 5 healthy controls have been isolated and compared by NGS with CD34+Hematopoietic stem and progenitors cells (HSPCs) for panel of 54 myeloid-associated mutations. PMF patients had higher levels of CECs. No mutation was found in HSPCs and CECs from controls, while CECs from PMF patients presented several somatic mutations. 72% of evaluable patients shared at least one mutation between HSPCs and CECs. 2 patients shared the JAK2 mutation, together with ABL1, IDH1, TET2 and ASXL1, KMT2A, respectively. 6 out of 8 shared only NON MPN-driver mutations: TET2 and NOTCH1 in one case; individual paired mutations in TP53, KIT, SRSF2, NOTCH1 and WT1, in the other cases. In conclusion, 70% of PMF patients shared at least one mutation between HSPCs and CECs. These latter harbored several myeloid-associated mutations, besides JAK2V617F mutation. Our results support a primary involvement of EC in PMF and provide a new methodological approach for further studies exploring the role of the "neoplastic" vascular niche.
Asunto(s)
Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mutación/genética , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patología , Anciano , Antígenos CD34/metabolismo , Estudios de Casos y Controles , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MasculinoRESUMEN
Regenerative medicine aims to restore damaged tissues and mainly takes advantage of human mesenchymal stromal cells (hMSCs), either alone or combined with three-dimensional scaffolds. The scaffold is generally considered a support, and its contribution to hMSC proliferation and differentiation is unknown or poorly investigated. The aim of this study was to evaluate the capability of an innovative three-dimensional gelatin-chitosan hybrid hydrogel scaffold (HC) to activate the osteogenic differentiation process in hMSCs. We seeded hMSCs from adipose tissue (AT-hMSCs) and bone marrow (BM-hMSCs) in highly performing HC of varying chitosan content in the presence of growing medium (GM) or osteogenic medium (OM) combined with Fetal Bovine Serum (FBS) or human platelet lysate (hPL). We primarily evaluated the viability and the proliferation of AT-hMSCs and BM-hMSCs under different conditions. Then, in order to analyse the activation of osteogenic differentiation, the osteopontin (OPN) transcript was absolutely quantified at day 21 by digital PCR. OPN was expressed under all conditions, in both BM-hMSCs and AT-hMSCs. Cells seeded in HC cultured with OM+hPL presented the highest OPN transcript levels, as expected. Interestingly, both BM-hMSCs and AT-hMSCs cultured with GM+FBS expressed OPN. In particular, BM-hMSCs cultured with GM+FBS expressed more OPN than those cultured with GM+hPL and OM+FBS; AT-hMSCs cultured with GM+FBS presented a lower expression of OPN when compared with those cultured with GM+hPL, but no significant difference was detected when compared with AT-hMSCs cultured with OM+FBS. No OPN expression was detected in negative controls. These results show the capability of HC to primarily and independently activate osteogenic differentiation pathways in hMCSs. Therefore, these scaffolds may be considered no more as a simple support, rather than active players in the differentiative and regenerative process.
RESUMEN
One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and -9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system.