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BACKGROUND: Enhancer reprogramming plays a significant role in the heterogeneity of cancer. However, we have limited knowledge about the impact of chromatin remodeling in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) patients, and how it affects tumorigenesis and drug response. Our research focuses on investigating the role of enhancers in sustaining oncogenic transformation in children with BCP-ALL. METHODS: We used ATAC-seq to study the accessibility of chromatin in pediatric BCP-ALL at three different stages-onset, remission, and relapse. Using a combination of computational and experimental methods, we were able to analyze the accessibility landscape and focus on the most significant cis-regulatory sites. These sites were then functionally validated through the use of Promoter capture Hi-C in a primary cell line model called LAL-B, followed by RNA-seq and genomic deletion of target sites using CRISPR-Cas9 editing. RESULTS: We found that enhancer activity changes during cancer progression and is mediated by the production of enhancer RNAs (eRNAs). CRISPR-Cas9-mediated validation of previously unknown eRNA productive enhancers demonstrated their capability to control the oncogenic activities of the MYB and DCTD genes. CONCLUSIONS: Our findings directly support the notion that productive enhancer engagement is a crucial determinant of the BCP-ALL and highlight the potential of enhancers as therapeutic targets in pediatric BCP-ALL.
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Transformação Celular Neoplásica , Progressão da Doença , Elementos Facilitadores Genéticos , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , CriançaRESUMO
Mucosal-associated invariant T (MAIT) cells are innate-like T-cells implicated in the response to fungal and bacterial infections. Their contribution to restoring T-cell immunity and influencing hematopoietic stem cell transplant (HSCT) outcomes remains poorly understood. We retrospectively studied MAIT-cell recovery in 145 consecutive children and young adults with hematological malignancies undergoing allo-HSCT, between April/2019 and May/2022, from unrelated matched donor (MUD, n=52), with standard graft-versus-host-disease (GvHD) prophylaxis, or HLA-haploidentical (Haplo, n=93) donor after in vitro αßT/CD19-cell depletion, without post-HSCT pharmacological prophylaxis. With a median follow-up of 33 months (12-49), overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM) were 79.5%, 72% and 7%, respectively; GvHD-free, Relapse-free Survival (GRFS) was 63%, while cumulative incidence of relapse was 23%. While WWT-cells reconstituted 1-2 years post-HSCT, MAIT-cells showed delayed recovery and prolonged functional impairment, characterized by expression of activation (CD25, CD38), exhaustion (PD1, TIM3) and senescence (CD57) markers, and suboptimal ex vivo response. OS, DFS and NRM were not affected by MAIT-cells. Interestingly, higher MAIT-cells at day+30 correlated with higher incidence of grade II-IV acute GvHD (19% vs 7%, p=0.06). Furthermore, a greater MAIT-cell count tended to be associated with a higher incidence of chronic GvHD (17% vs 6%, p=0.07) resulting in lower GRFS (55% vs 73%, p=0.05). Higher MAIT-cells also correlated with greater cytomegalovirus (CMV) reactivation and lower late blood stream infections (BSI) (44% vs 24%, p=0.02 and 9% vs 18%, p=0.08, respectively). Future studies are needed to confirm the impact of early MAIT-cell recovery on cGvHD, CMV reactivation and late BSI.
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ABSTRACT: TCRαß/CD19 cell depletion is a promising graft manipulation technique frequently used in the context of human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation (HSCT). We previously reported the results of a phase I-II clinical trial (NCT01810120) to assess the safety and the efficacy of this type of exvivo T-cell depletion in 80 children with acute leukemia, showing promising survival outcomes. We now report an updated analysis on a cohort of 213 children with a longer follow-up (median, 47.6 months for surviving patients). With a 5-year cumulative incidence of nonrelapse mortality of 5.2% (95% confidence interval [CI], 2.8%-8.8%) and a cumulative incidence of relapse of 22.7% (95% CI, 16.9%-29.2%), projected 10-year overall and disease-free survival (DFS) were 75.4% (95% CI, 68.6%-80.9%) and 71.6% (95% CI, 64.4%-77.6%), respectively. Cumulative incidence of both grade II-IV acute and chronic graft-versus-host disease were low (14.7% and 8.1%, respectively). In a multivariable analysis for DFS including type of disease, use of total body irradiation in the conditioning regimen (hazard ratio [HR], 0.5; 95% CI, 0.26-0.98; P = .04), disease status at HSCT (complete remission [CR] ≥3 vs CR 1/2; HR, 2.23; 95% CI, 1.20-4.16; P = .01), and high levels of pre-HSCT minimal residual disease (HR, 2.09; 95% CI, 1.01-4.33; P = .04) were independently associated with outcome. In summary, besides confirming the good outcome results already reported (which are almost superimposable on those of transplant from HLA-matched donors), this clinical update allows the identification of patients at higher risk of treatment failure for whom personalized approaches, aimed at reducing the risk of relapse, are warranted.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Criança , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta , Transplante Haploidêntico/efeitos adversos , Antígenos HLA , Transplante de Células-Tronco Hematopoéticas/métodos , Antígenos de Histocompatibilidade Classe II , Recidiva , Condicionamento Pré-Transplante/métodos , Estudos RetrospectivosRESUMO
Autologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) or displaying profound lymphopenia and/or rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells. We tested donor-derived T cells transduced with a second-generation (4.1BB) CD19-directed CAR for treatment of patients with BCP-ALL in a hospital-exemption setting. Two constructs were tested: a retroviral construct incorporating the suicide gene inducible caspase-9 (CD19-CAR-Retro_ALLO) first and then a lentiviral construct and an automated, Prodigy-based manufacturing process (CD19-CAR-Lenti_ALLO). Thirteen children/young adults received ALLO-CAR-T cells between March 2021 and October 2022. Doses ranged between 1.0 × 106 and 3.0 × 106 CAR-T cells per kg. The toxicity profile was comparable with that of autologous CAR-T cells, characterized mainly by cytopenia, cytokine release syndrome (maximum grade 1), and grade 2 immune-effector cell-associated neurotoxicity syndrome. One case of acute graft-versus-host disease (GVHD) occurred and was rapidly controlled with steroids and ruxolitinib. None of the other patients, including 3 given ALLO-CAR-T cells from an HLA-haploidentical donor, experienced GVHD. Two patients received ALLO-CAR-T cells before HSCT and showed a significant expansion of CAR-T cells without any sign of GVHD. All patients obtained complete remission (CR) with absence of minimal residual disease in the bone marrow. With a median follow-up of 12 months (range, 5-21), 8 of 13 patients maintained CR. Allogeneic anti-CD19 CAR-T cells can effectively treat highly refractory BCP-ALL relapsing after allo-HSCT without showing increased toxicity as compared with autologous CAR-T cells.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto Jovem , Humanos , Criança , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linfócitos T , Doença Enxerto-Hospedeiro/etiologia , Imunoterapia Adotiva/efeitos adversos , Antígenos CD19RESUMO
BACKGROUND: Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma. METHODS: In an academic, phase 1-2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01). RESULTS: A total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×106 CAR-positive T cells per kilogram of body weight) in the phase 1 portion of the trial, and no dose-limiting toxic effects were recorded; the recommended dose for the phase 2 portion of the trial was 10×106 CAR-positive T cells per kilogram. Cytokine release syndrome occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion (median persistence, 3 months; range, 1 to 30). Seventeen children had a response to the treatment (overall response, 63%); 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year overall survival and event-free survival were 60% and 36%, respectively. CONCLUSIONS: The use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT03373097.).
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Imunoterapia Adotiva , Neuroblastoma , Receptores de Antígenos Quiméricos , Criança , Humanos , Caspase 9/efeitos adversos , Caspase 9/genética , Caspase 9/metabolismo , Caspase 9/uso terapêutico , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/terapia , Neuroblastoma/genética , Neuroblastoma/terapia , Receptores de Antígenos Quiméricos/uso terapêuticoRESUMO
Hematopoietic stem cell transplantation (HSCT)-based approaches are increasingly investigated strategies to induce tolerance in recipients of solid allografts. However, in the majority of cases, these approaches rely on the infusion of hematopoietic stem cells recovered from the same solid organ donor. In this report, we describe the case of a boy who received liver transplantation from a deceased donor, who had successfully underwent allogeneic HSCT from an unrelated donor for hepatitis-associated aplastic anemia. In this patient, it was possible to permanently withdraw post-HSCT immune suppression without causing any sign of liver graft dysfunction. To the best of our knowledge, this is the first case of operational tolerance documented in a patient who received combined liver transplantation and HSCT from different donors.
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Anemia Aplástica , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Transplante de Fígado , Masculino , Humanos , Criança , Transplante de Fígado/efeitos adversos , Doadores de Tecidos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Tolerância Imunológica , Transplante Homólogo/efeitos adversos , Anemia Aplástica/etiologia , Doença Enxerto-Hospedeiro/etiologiaRESUMO
CD8+ T-cell activation has been demonstrated to distinguish patients with primary and infection-associated hemophagocytic lymphohistiocytosis (HLH) from patients with early sepsis. We evaluated the activation profile of CD8+ T cells in patients with various forms of secondary HLH (sHLH), including macrophage activation syndrome (MAS). Peripheral blood mononuclear cells from children with inactive systemic juvenile idiopathic arthritis (sJIA, n = 17), active sJIA (n = 27), MAS in sJIA (n = 14), infection-associated HLH (n = 7), and with other forms of sHLH (n = 9) were analyzed by flow cytometry. Compared with patients with active sJIA, in patients with MAS and sHLH of different origins, beside a significant increase in the frequency of CD38high/HLA-DR+CD8+ T cells, we found a significant increase in the frequency of CD8+ T cells expressing the CD4 antigen (CD4dimCD8+ T cells). These cells expressed high levels of the activation markers CD38 and HLA-DR, suggesting they were a subset of CD38high/HLA-DR+CD8+ T cells, as well as of the activation/exhaustion markers CD25, PD1, CD95, and interferon-γ. The frequency of CD4dimCD8+ T cells strongly correlated with most of the laboratory parameters of MAS severity and with circulating levels of CXCL9 and interleukin-18. These findings were confirmed in a prospective replication cohort in which no expansion of any particular T-cell receptor Vß family in CD3+ T cells of patients with sHLH was found. Finally, frequency of CD4dimCD8+, but not of CD38high/HLA-DR+CD8+ T cells, significantly correlated with a clinical severity score, further supporting the involvement of these cells in MAS/sHLH pathogenesis.
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Artrite Juvenil , Linfo-Histiocitose Hemofagocítica , Síndrome de Ativação Macrofágica , Artrite Juvenil/complicações , Criança , Humanos , Leucócitos Mononucleares/patologia , Linfo-Histiocitose Hemofagocítica/patologia , Síndrome de Ativação Macrofágica/patologia , Estudos ProspectivosRESUMO
Several diseases are associated with alterations of the B-cell compartment. Knowing how to correctly identify by flow cytometry the distribution of B-cell populations in the peripheral blood is important to help in the early diagnosis. In the accompanying article we describe how to identify the different B-cell subsets in the peripheral blood of healthy donors. Here we show a few examples of diseases that cause dysregulation of the B-cell compartment.
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Subpopulações de Linfócitos B , Linfócitos B , Citometria de Fluxo , HumanosRESUMO
Several nonmalignant disorders (NMDs), either inherited or acquired, can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). Between January 2012 and April 2020, 70 consecutive children affected by primary immunodeficiencies, inherited/acquired bone marrow failure syndromes, red blood cell disorders, or metabolic diseases, lacking a fully matched donor or requiring urgent transplantation underwent TCRαß/CD19-depleted haploidentical HSCT from an HLA-partially matched relative as part of a prospective study. The median age at transplant was 3.5 years (range 0.3-16.1); the median time from diagnosis to transplant was 10.5 months (2.7 for SCID patients). Primary engraftment was obtained in 51 patients, while 19 and 2 patients experienced either primary or secondary graft failure (GF), the overall incidence of this complication being 30.4%. Most GFs were observed in children with disease at risk for this complication (eg, aplastic anemia, thalassemia). All but 5 patients experiencing GF were successfully retransplanted. Six patients died of infectious complications (4 had active/recent infections at the time of HSCT), the cumulative incidence of transplant-related mortality (TRM) being 8.5%. Cumulative incidence of grade 1-2 acute GVHD was 14.4% (no patient developed grade 3-4 acute GVHD). Only one patient at risk developed mild chronic GVHD. With a median follow-up of 3.5 years, the 5-year probability of overall and disease-free survival was 91.4% and 86.8%, respectively. In conclusion, TCRαß/CD19-depleted haploidentical HSCT from an HLA-partially matched relative is confirmed to be an effective treatment of children with NMDs. Prompt donor availability, low incidence of GVHD, and TRM make this strategy an attractive option in NMDs patients. The study is registered at ClinicalTrial.gov as NCT01810120.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Adolescente , Criança , Pré-Escolar , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Lactente , Estudos Prospectivos , Receptores de Antígenos de Linfócitos T alfa-beta , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
The outcome of patients affected by high-risk or metastatic neuroblastoma (NB) remains grim, with ≥ 50% of the children experiencing relapse or progression of the disease despite multimodal, intensive treatment. In order to identify new strategies to improve the overall survival and the quality of life of these children, we recently developed and optimized a third-generation GD2-specific chimeric antigen receptor (CAR) construct, which is currently under evaluation in our Institution in a phase I/II clinical trial (NCT03373097) enrolling patients with relapsed/refractory NB. We observed that our CAR T-cells are able to induce marked tumor reduction and even achieve complete remission with a higher efficiency than that of other CAR T-cells reported in previous studies. However, often responses are not sustained and relapses occur. Here, we demonstrate for the first time a mechanism of resistance to GD2.CAR T-cell treatment, showing how polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) increase in the peripheral blood (PB) of NB patients after GD2.CAR T-cell treatment in case of relapse and loss of response. In vitro, isolated PMN-MDSC demonstrate to inhibit the anti-tumor cytotoxicity of different generations of GD2.CAR T-cells. Gene-expression profiling of GD2.CAR T-cells "conditioned" with PMN-MDSC shows downregulation of genes involved in cell activation, signal transduction, inflammation and cytokine/chemokine secretion. Analysis of NB gene-expression dataset confirms a correlation between expression of these genes and patient outcome. Moreover, in patients treated with GD2.CAR T-cells, the frequency of circulating PMN-MDSC inversely correlates with the levels of GD2.CAR T-cells, resulting more elevated in patients who did not respond or lost response to the treatment. The presence and the frequency of PMN-MDSC in PB of high-risk and metastatic NB represents a useful prognostic marker to predict the response to GD2.CAR T-cells and other adoptive immunotherapy. This study underlines the importance of further optimization of both CAR T-cells and clinical trial in order to target elements of the tumor microenvironment.
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Imunoterapia Adotiva , Células Supressoras Mieloides/imunologia , Neuroblastoma/terapia , Humanos , Imunoterapia Adotiva/métodos , Células Supressoras Mieloides/patologia , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Neuroblastoma/imunologia , Neuroblastoma/patologia , Resultado do TratamentoRESUMO
Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. BACKGROUND: METHODS: We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. RESULTS: The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. CONCLUSIONS: Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.
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Epitopos/imunologia , Leucemia de Células B/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
We report on the outcome of 24 patients with Fanconi anemia (FA) lacking an HLA matched related or unrelated donor, given an HLA-haploidentical T-cell receptor αß (TCRαß+) and CD19+ cell-depleted hematopoietic stem cell transplantation (HSCT) in the context of a prospective, single-center phase 2 trial. Sustained primary engraftment was achieved in 22 (91.6%) of 24 patients, with median time to neutrophil recovery of 12 days (range, 9-15 days) and platelet recovery of 10 days (range, 7-14 days). Cumulative incidences of grade 1 to 2 acute graft-versus-host disease (GVHD) and chronic GVHD were 17.4% (95% confidence interval [CI], 5.5%-35.5%) and 5.5% (95% CI, 0.8%-33.4%), respectively. The conditioning regimen, which included fludarabine, low-dose cyclophosphamide and, in most patients, single-dose irradiation was well tolerated; no fatal transplant-related toxicity was observed. With a median follow-up of 5.2 years (range, 0.3-8.7 years), the overall and event-free survival probabilities were 100% and 86.3% (95% CI, 62.8%-95.4%), respectively (2 graft failures and 1 case of poor graft function were considered as events). The 2 patients who experienced primary graft failure underwent a subsequent successful HSCT from the other parent. This is the first report of FA patients given TCRαß+/CD19+-depleted haplo-HSCT in the context of a prospective trial, and the largest series of T-cell-depleted haplo-HSCT in FA reported to date. This trial was registered at www.clinicaltrials.gov as #NCT01810120.
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Anemia de Fanconi , Transplante de Células-Tronco Hematopoéticas , Criança , Anemia de Fanconi/terapia , Humanos , Estudos Prospectivos , Receptores de Antígenos de Linfócitos T alfa-beta , Condicionamento Pré-Transplante , Adulto JovemRESUMO
TcRαß/CD19-cell depleted HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) represents a promising new platform for children affected by acute leukemia in need of an allograft and lacking a matched donor, disease recurrence being the main cause of treatment failure. The use of zoledronic acid to enhance TcRγδ+ lymphocyte function after TcRαß/CD19-cell depleted haplo-HSCT was tested in an open-label, feasibility, proof-of-principle study. Forty-six children affected by high-risk acute leukemia underwent haplo-HSCT after removal of TcRαß+ and CD19+ B lymphocytes. No post-transplant pharmacological graft-versus-host disease (GvHD) prophylaxis was given. Zoledronic acid was administered monthly at a dose of 0.05 mg/kg/dose (maximum dose 4 mg), starting from day +20 after transplantation. A total of 139 infusions were administered, with a mean of 3 infusions per patient. No severe adverse event was observed. Common side effects were represented by asymptomatic hypocalcemia and acute phase reactions (including fever, chills, malaise, and/or arthralgia) within 24-48 h from zoledronic acid infusion. The cumulative incidence of acute and chronic GvHD was 17.3% (all grade I-II) and 4.8% (all limited), respectively. Patients given 3 or more infusions of zoledronic acid had a lower incidence of both acute GvHD (8.8 vs. 41.6%, p = 0.015) and chronic GvHD (0 vs. 22.2%, p = 0.006). Transplant-related mortality (TRM) and relapse incidence at 3 years were 4.3 and 30.4%, respectively. Patients receiving repeated infusions of zoledronic acid had a lower TRM as compared to those receiving 1 or 2 administration of the drug (0 vs. 16.7%, p = 0.01). Five-year overall survival (OS) and disease-free survival (DFS) for the whole cohort were 67.2 and 65.2%, respectively, with a trend toward a better OS for patients receiving 3 or more infusions (73.1 vs. 50.0%, p = 0.05). The probability of GvHD/relapse-free survival was significantly worse in patients receiving 1-2 infusions of zoledonic acid than in those given ≥3 infusions (33.3 vs. 70.6%, respectively, p = 0.006). Multivariable analysis showed an independent positive effect on outcome given by repeated infusions of zoledronic acid (HR 0.27, p = 0.03). These data indicate that the use of zoledronic acid after TcRαß/CD19-cell depleted haploHSCT is safe and may result in a lower incidence of acute GvHD, chronic GvHD, and TRM.
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Antígenos CD19/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Fatores Imunológicos/administração & dosagem , Leucemia Mieloide Aguda/terapia , Depleção Linfocítica/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/efeitos dos fármacos , Condicionamento Pré-Transplante/métodos , Transplante Haploidêntico/métodos , Ácido Zoledrônico/administração & dosagem , Adolescente , Linfócitos B/imunologia , Criança , Pré-Escolar , Estudos de Coortes , Intervalo Livre de Doença , Estudos de Viabilidade , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Fatores Imunológicos/efeitos adversos , Lactente , Masculino , Linfócitos T/imunologia , Transplante Homólogo/métodos , Adulto Jovem , Ácido Zoledrônico/efeitos adversosRESUMO
Traditionally, hematopoietic stem cell transplantation (HSCT) from both HLA-matched related and unrelated donors (UD) has been used for treating children with acute leukemia (AL) in need of an allograft. Recently, HLA-haploidentical HSCT after αß T-cell/B-cell depletion (αßhaplo-HSCT) was shown to be effective in single-center studies. Here, we report the first multicenter retrospective analysis of 127 matched UD (MUD), 118 mismatched UD (MMUD), and 98 αßhaplo-HSCT recipients, transplanted between 2010 and 2015, in 13 Italian centers. All these AL children were transplanted in morphological remission after a myeloablative conditioning regimen. Graft failure occurred in 2% each of UD-HSCT and αßhaplo-HSCT groups. In MUD vs MMUD-HSCT recipients, the cumulative incidence of grade II to IV and grade III to IV acute graft-versus-host disease (GVHD) was 35% vs 44% and 6% vs 18%, respectively, compared with 16% and 0% in αßhaplo-HSCT recipients (P < .001). Children treated with αßhaplo-HSCT also had a significantly lower incidence of overall and extensive chronic GVHD (P < .01). Eight (6%) MUD, 32 (28%) MMUD, and 9 (9%) αßhaplo-HSCT patients died of transplant-related complications. With a median follow-up of 3.3 years, the 5-year probability of leukemia-free survival in the 3 groups was 67%, 55%, and 62%, respectively. In the 3 groups, chronic GVHD-free/relapse-free (GRFS) probability of survival was 61%, 34%, and 58%, respectively (P < .001). When compared with patients given MMUD-HSCT, αßhaplo-HSCT recipients had a lower cumulative incidence of nonrelapse mortality and a better GRFS (P < .001). These data indicate that αßhaplo-HSCT is a suitable therapeutic option for children with AL in need of transplantation, especially when an allele-matched UD is not available.
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Linfócitos B , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia , Depleção Linfocítica , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T , Doadores não Relacionados , Doença Aguda , Adolescente , Aloenxertos , Criança , Pré-Escolar , Doença Crônica , Feminino , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Lactente , Leucemia/mortalidade , Leucemia/terapia , Masculino , Estudos RetrospectivosRESUMO
Despite progress in treating B-cell precursor acute lymphoblastic leukemia (BCP-ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high-risk relapsed patients. Che-1/AATF (Che-1) is an RNA polymerase II-binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che-1 is overexpressed in pediatric BCP-ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP-ALL cells. Furthermore, we report that c-Myc regulates Che-1 expression by direct binding to its promoter and describe a strict correlation between Che-1 expression and c-Myc expression. RNA-seq analyses upon Che-1 or c-Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP-seq experiments suggest that Che-1 acts as a downstream effector of c-Myc. These results identify the pivotal role of Che-1 in the control of BCP-ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP-ALL.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas/genéticaRESUMO
Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children with acute leukemia (AL) either relapsed or at high-risk of treatment failure. We developed a novel method of graft manipulation based on negative depletion of αß T and B cells and conducted a prospective trial evaluating the outcome of children with AL transplanted with this approach. Eighty AL children, transplanted between September 2011 and September 2014, were enrolled in the trial. All children were given a fully myeloablative preparative regimen. Anti-T-lymphocyte globulin from day -5 to -3 was used for preventing graft rejection and graft-versus-host disease (GVHD); no patient received any posttransplantation GVHD prophylaxis. Two children experienced primary graft failure. The cumulative incidence of skin-only, grade 1-2 acute GVHD was 30%; no patient developed extensive chronic GVHD. Four patients died, the cumulative incidence of nonrelapse mortality being 5%, whereas 19 relapsed, resulting in a 24% cumulative incidence of relapse. With a median follow-up of 46 months for surviving patients, the 5-year probability of chronic GVHD-free, relapse-free survival (GRFS) is 71%. Total body irradiation-containing preparative regimen was the only variable favorably influencing relapse incidence and GRFS. The outcomes of these 80 patients are comparable to those of 41 and 51 children given transplantation from an HLA-identical sibling or a 10/10 allelic-matched unrelated donor in the same period. These data indicate that haplo-HSCT after αß T- and B-cell depletion represents a competitive alternative for children with AL in need of urgent allograft. This trial was registered at www.clinicaltrials.gov as #NCT01810120.
Assuntos
Linfócitos B , Transplante de Células-Tronco Hematopoéticas , Leucemia , Depleção Linfocítica , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T , Doença Aguda , Adolescente , Adulto , Aloenxertos , Soro Antilinfocitário/administração & dosagem , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Lactente , Leucemia/mortalidade , Leucemia/terapia , Masculino , Taxa de SobrevidaRESUMO
Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αß T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αß T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αß T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αß T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.
RESUMO
Achieving complete remission (CR) in childhood relapsed/refractory acute lymphoblastic leukaemia (ALL) is a difficult task. Bortezomib, a proteasome inhibitor, has in vitro activity against ALL blasts. A phase I-II trial, reported by the Therapeutic Advances in Childhood Leukaemia and Lymphoma (TACL) consortium, demonstrated that bortezomib with chemotherapy has acceptable toxicity and remarkable activity in patients with relapsed ALL failing 2-3 previous regimens. We evaluated bortezomib in combination with chemotherapy in 30 and 7 children with B-cell precursor (BCP) and T-cell ALL, respectively. Bortezomib (1·3 mg/m2 /dose) was administered intravenously on days 1, 4, 8, and 11. Chemotherapy agents were the same as those used in the TACL trial, consisting of dexamethasone, doxorubicin, vincristine and pegylated asparaginase. Three patients (8·1%) died due to infections. Twenty-seven patients (72·9%) achieved CR or CR with incomplete platelet recovery (CRp). Fourteen had minimal residual disease (MRD) lower than 0·1%. Twenty-two of 30 BCP-ALL patients (73·3%) and 5/7 patients (71%) with T-cell ALL achieved CR/CRp. The 2-year overall survival (OS) is 31·3%; CR/CRp patients with an MRD response had a remarkable 2-year OS of 68·4%. These data confirm that the combination of bortezomib with chemotherapy is a suitable/effective option for childhood relapsed/refractory ALL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bortezomib/administração & dosagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Asparaginase , Criança , Pré-Escolar , Dexametasona , Doxorrubicina , Feminino , Humanos , Lactente , Masculino , Neoplasia Residual , Polietilenoglicóis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Inibidores de Proteassoma , Indução de Remissão , Terapia de Salvação/métodos , Análise de Sobrevida , Vincristina , Adulto JovemRESUMO
INTRODUCTION: Cytomegalovirus (CMV) still causes significant morbidity and mortality in patients given allogeneic hematopoietic stem cell transplantation (HSCT). Despite effective pharmacotherapy, potentially life-threatening CMV disease occurs nowadays in up to 10% of HSCT recipients; moreover, routinely used anti-CMV agents have been shown to be associated with morbidity. Areas covered: This review examines different issues related to diagnosis and management of CMV infection in HSCT recipients, paying particular attention to the monitoring of CMV-specific immune recovery, approaches of adoptive cell therapy and new antiviral drugs. Expert commentary: Despite advances in diagnostic tests and treatment, there is still room for refining management of CMV in HSCT recipients. Immunological monitoring should be associated in the future to virological monitoring. The safety profile and efficacy of new anti-CMV agents should be compared with that of standard-of-care drugs. Donor-derived, pathogen-specific T cells adoptively transferred after transplantation could contribute to reduce the impact of CMV infection on patient's outcome.