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BACKGROUND AND AIMS: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) improves the prognosis in pediatric patients with several solid tumors and lymphomas. Little is known about the reconstitution of the immune system after ASCT and the influence of CD34+ cell selection on the reconstitution in pediatric patients. METHODS: Between 1990 and 2001, 94 pediatric patients with solid tumors and lymphomas received autologous CD34+ selected or unmanipulated peripheral stem cells after HDC. CD34+ selection was carried out with magnetic microbeads. The absolute numbers of T cells, B cells and natural killer (NK) cells were measured and compared in both groups at various time points post-transplant. RESULTS: Recovery of T cells was significantly faster in the unmanipulated group at day 30, with no significant difference later on. Reconstitution of B and NK cells was similar in both groups without significant differences at any time. The CD34+-selected group was divided into patients receiving less or more than 5.385 × 106/kg CD34+ cells. Patients in the CD34+ high-dose group displayed significantly faster reconstitutions of neutrophiles and lymphocyte subsets than the CD34+ low-dose group. CONCLUSIONS: Engraftment and reconstitution of leukocytes, B cells and NK cells after transplantation of CD34+ selected stem cells were comparable to that in patients receiving unmanipulated grafts. T-cell recovery was faster in the unmanipulated group only within the first month. However, this delay could be compensated by transplantation of >5.385 × 106 CD34+ cells/kg. Especially for patients receiving immunotherapy after HDC large numbers of immune effector cells such as NK and T cells are necessary to mediate antibody-dependent cellular cytotoxicity. Therefore, in patients receiving autologous CD34+-selected grafts, our data emphasize the need to administer high stem cell counts.
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BACKGROUND: Neuroblastoma is the most common solid tumor in infants accounting for approximately 15% of all cancer-related deaths. Over 50% of high-risk neuroblastoma relapse, emphasizing the need of novel drug targets and therapeutic strategies. In neuroblastoma, chromosomal gains at chromosome 17q, including IGF2BP1, and MYCN amplification at chromosome 2p are associated with adverse outcome. Recent, pre-clinical evidence indicates the feasibility of direct and indirect targeting of IGF2BP1 and MYCN in cancer treatment. METHODS: Candidate oncogenes on 17q were identified by profiling the transcriptomic/genomic landscape of 100 human neuroblastoma samples and public gene essentiality data. Molecular mechanisms and gene expression profiles underlying the oncogenic and therapeutic target potential of the 17q oncogene IGF2BP1 and its cross-talk with MYCN were characterized and validated in human neuroblastoma cells, xenografts and PDX as well as novel IGF2BP1/MYCN transgene mouse models. RESULTS: We reveal a novel, druggable feedforward loop of IGF2BP1 (17q) and MYCN (2p) in high-risk neuroblastoma. This promotes 2p/17q chromosomal gains and unleashes an oncogene storm resulting in fostered expression of 17q oncogenes like BIRC5 (survivin). Conditional, sympatho-adrenal transgene expression of IGF2BP1 induces neuroblastoma at a 100% incidence. IGF2BP1-driven malignancies are reminiscent to human high-risk neuroblastoma, including 2p/17q-syntenic chromosomal gains and upregulation of Mycn, Birc5, as well as key neuroblastoma circuit factors like Phox2b. Co-expression of IGF2BP1/MYCN reduces disease latency and survival probability by fostering oncogene expression. Combined inhibition of IGF2BP1 by BTYNB, MYCN by BRD inhibitors or BIRC5 by YM-155 is beneficial in vitro and, for BTYNB, also. CONCLUSION: We reveal a novel, druggable neuroblastoma oncogene circuit settling on strong, transcriptional/post-transcriptional synergy of MYCN and IGF2BP1. MYCN/IGF2BP1 feedforward regulation promotes an oncogene storm harboring high therapeutic potential for combined, targeted inhibition of IGF2BP1, MYCN expression and MYCN/IGF2BP1-effectors like BIRC5.
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Neuroblastoma , Animais , Humanos , Lactente , Camundongos , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Recidiva Local de Neoplasia/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismoRESUMO
BACKGROUND: Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse. METHODS: ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively. RESULTS: At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients <18 months. At relapse, ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome. CONCLUSIONS: The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.
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Neuroblastoma , Receptores Proteína Tirosina Quinases , Criança , Humanos , Quinase do Linfoma Anaplásico/genética , Receptores Proteína Tirosina Quinases/genética , Recidiva Local de Neoplasia/genética , Neuroblastoma/genética , Neuroblastoma/patologia , GenômicaRESUMO
Therapy-resistant viral reactivations contribute significantly to mortality after hematopoietic stem cell transplantation. Adoptive cellular therapy with virus-specific T cells (VST) has shown efficacy in various single-center trials. However, the scalability of this therapy is hampered by laborious production methods. In this study we describe the in-house production of VST in a closed system (CliniMACS Prodigy® system, Miltenyi Biotec). In addition, we report the efficacy in 26 patients with viral disease following hematopoietic stem cell transplantation in a retrospective analysis (adenovirus, n=7; cytomegalovirus, n=8; Epstein-Barr virus, n=4; multi-viral, n=7). The production of VST was successful in 100% of cases. The safety profile of VST therapy was favorable (n=2 grade 3 and n=1 grade 4 adverse events; all three were reversible). A response was seen in 20 of 26 patients (77%). Responding patients had a significantly better overall survival than patients who did not respond (P<0.001). Virus-specific symptoms were reduced or resolved in 47% of patients. The overall survival of the whole cohort was 28% after 6 months. This study shows the feasibility of automated VST production and safety of application. The scalability of the CliniMACS Prodigy® device increases the accessibility of VST treatment.
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Infecções por Vírus Epstein-Barr , Transplante de Células-Tronco Hematopoéticas , Viroses , Humanos , Linfócitos T , Infecções por Vírus Epstein-Barr/terapia , Estudos Retrospectivos , Herpesvirus Humano 4 , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Viroses/etiologia , Viroses/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-TroncoRESUMO
BACKGROUND: In many organisms, including humans, the timing of cellular processes is regulated by the circadian clock. At the molecular level the core-clock consists of transcriptional-translational-feedback loops including several genes such as BMAL1, CLOCK, PERs and CRYs generating circa 24-h rhythms in the expression of about 40% of our genes across all tissues. Previously these core-clock genes have been shown to be differentially expressed in various cancers. Albeit a significant effect in treatment optimization of chemotherapy timing in paediatric acute lymphoblastic leukaemia has previously been reported, the mechanistic role played by the molecular circadian clock in acute paediatric leukaemia remains elusive. METHODS: To characterize the circadian clock, we will recruit patients with newly diagnosed leukaemia and collect time course saliva and blood samples, as well as a single bone marrow sample. From the blood and bone marrow samples nucleated cells will be isolated and further undergo separation into CD19+ and CD19- cells. qPCR is performed on all samples targeting the core-clock genes including BMAL1, CLOCK, PER2 and CRY1. Resulting data will be analysed for circadian rhythmicity using the RAIN algorithm and harmonic regression. DISCUSSION: To the best of our knowledge this is the first study aiming to characterize the circadian clock in a cohort of paediatric patients with acute leukaemia. In the future we hope to contribute to uncovering further vulnerabilities of cancers associated with the molecular circadian clock and in particular adjust chemotherapy accordingly, leading to more targeted toxicity, and hence decreased systemic toxicities.
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Relógios Circadianos , Leucemia , Humanos , Criança , Estudos Prospectivos , Fatores de Transcrição ARNTL , Proteínas Adaptadoras de Transdução de SinalRESUMO
Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma.
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Retroalimentação , Modelos Biológicos , Neuroblastoma/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Sistema de Sinalização das MAP Quinases , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismoRESUMO
In the rare co-occurrence of childhood cancer and severe hemophilia, hemostatic management is of paramount therapeutic importance. We present the case of an 11-month-old boy with severe congenital hemophilia B, who was diagnosed with metastatic high-risk neuroblastoma. He consequently developed paraneoplastic coagulopathy with life-threatening tumor hemorrhage and intracranial hemorrhage, showing central nervous system relapse. Management consisted of factor IX replacement with extended half-life factor IX fusion protein, adjusted to bleeding risk. Additional interventions included factor XIII, fibrinogen, fresh frozen plasma, tranexamic acid, and platelet transfusions. The half-life of factor IX products was markedly reduced requiring close factor IX monitoring and adequate replacement. This intensified treatment allowed chemotherapy, autologous stem cell transplantation, and GD2 antibody immune therapy without bleeding or thrombosis.
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Fator IX/administração & dosagem , Hemofilia B , Hemostáticos/administração & dosagem , Neuroblastoma , Proteínas Recombinantes de Fusão/administração & dosagem , Transplante de Células-Tronco , Neoplasias Abdominais/sangue , Neoplasias Abdominais/diagnóstico por imagem , Neoplasias Abdominais/terapia , Autoenxertos , Fator IX/farmacocinética , Hemofilia B/sangue , Hemofilia B/diagnóstico por imagem , Hemofilia B/terapia , Humanos , Lactente , Masculino , Neuroblastoma/sangue , Neuroblastoma/diagnóstico por imagem , Neuroblastoma/terapia , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
BACKGROUND: Several paediatric malignancies, including anaplastic large cell lymphoma (ALCL), inflammatory myofibroblastic tumour (IMT), neuroblastoma, and rhabdomyosarcoma, harbour activation of anaplastic lymphoma kinase (ALK) through different mechanisms. Here, we report the safety, pharmacokinetics, and efficacy of ceritinib in paediatric patients with ALK-positive malignancies. METHODS: This multicentre, open-label, phase 1 trial was done at 23 academic hospitals in ten countries. Children (aged ≥12 months to <18 years) diagnosed with locally advanced or metastatic ALK-positive malignancies that had progressed despite standard therapy, or for which no effective standard therapy were available, were eligible. ALK-positive malignancies were defined as those with ALK rearrangement, amplification, point mutation, or in the case of rhabdomyosarcoma, expression in the absence of any genetic alteration. Eligible patients had evaluable or measurable disease as defined by either Response Evaluation Criteria in Solid Tumours, version 1.1 for patients with non-haematological malignancies, International Neuroblastoma Response Criteria scan for patients with neuroblastoma, or International Working Group criteria for patients with lymphoma. Other eligibility criteria were Karnofsky performance status score of at least 60% for patients older than 12 years or Lansky score of at least 50% for patients aged 12 years or younger. This study included a dose-escalation part, followed by a dose-expansion part, in which all patients received treatment at the recommended dose for expansion (RDE) established in the dose-escalation part. Both parts of the study were done in fasted and fed states. In the dose-escalation part, patients were treated with once-daily ceritinib orally, with dose adjusted for body-surface area, rounded to the nearest multiple of the 50 mg dose strength. The starting dose in the fasted state was 300 mg/m2 daily and for the fed state was 320 mg/m2 daily. The primary objective of this study was to establish the maximum tolerated dose (ie, RDE) of ceritinib in the fasted and fed states. The RDE was established on the basis of the incidence of dose-limiting toxicities in patients who completed a minimum of 21 days of treatment with safety assessments and at least 75% drug exposure, or who discontinued treatment earlier because of dose-limiting toxicity. Overall response rate (defined as the proportion of patients with a best overall response of complete response or partial response) was a secondary endpoint. Activity and safety analyses were done in all patients who received at least one dose of ceritinib. This trial is registered with ClinicalTrials.gov (NCT01742286) and is completed. FINDINGS: Between Aug 28, 2013, and Oct 17, 2017, 83 children with ALK-positive malignancies were enrolled to the dose-escalation (n=40) and dose-expansion (n=43) groups. The RDE of ceritinib was established as 510 mg/m2 (fasted) and 500 mg/m2 (fed). 55 patients (30 with neuroblastoma, ten with IMT, eight with ALCL, and seven with other tumour types) were treated with ceritinib at the RDE (13 patients at 510 mg/m2 fasted and 42 patients at 500 mg/m2 fed). The median follow-up was 33·3 months (IQR 24·8-39·3) for patients with neuroblastoma, 33·2 months (27·9-35·9) for those with IMT, 34·0 months (21·9-46·4) for those with ALCL, and 27·5 months (22·4-36·9) for patients with other tumour types. An overall response was recorded in six (20%; 95% CI 8-39) of 30 patients with neuroblastoma, seven (70%; 33-93) of ten patients with IMT, six (75%; 35-97) of eight patients with ALCL, and one (14%; <1-58) of seven patients with other tumours. The safety profile of ceritinib was consistent with that observed in adult patients. All patients had at least one adverse event. Grade 3 or 4 adverse events occurred in 67 (81%) of 83 patients and were mostly increases in aminotransferases (alanine aminotransferase increase in 38 [46%] patients and aspartate aminotransferase increase in 27 [33%] patients). At least one serious adverse event was reported in 40 (48%) of 83 patients and 31 (37%) of 83 patients had at least one grade 3 or 4 serious adverse event. 14 (17%) deaths occurred during the study, of which 12 were on-treatment deaths and two were after 30 days of the last dose. Of the 12 on-treatment deaths, ten were due to disease progression (neuroblastoma), one due to sepsis, and one due to intractable hypotension. INTERPRETATION: Ceritinib 500 mg/m2 once daily with food is the recommended dose for paediatric patients with ALK-positive malignancies. Ceritinib showed promising preliminary antitumour activity in patients with ALK-positive refractory or recurrent IMT or ALCL, and in a subset of patients with relapsed or refractory neuroblastoma, with a manageable safety profile. Our data support the notion that ALK inhibitors should be considered in therapeutic strategies for paediatric patients with malignancies with genetic ALK alterations. FUNDING: Novartis Pharmaceutical Corporation.
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Quinase do Linfoma Anaplásico/metabolismo , Neoplasias/tratamento farmacológico , Pirimidinas/uso terapêutico , Sulfonas/uso terapêutico , Adolescente , Quinase do Linfoma Anaplásico/genética , Antineoplásicos/uso terapêutico , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Masculino , Dose Máxima Tolerável , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Taxa de SobrevidaRESUMO
Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing 13 C-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.
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Amplificação de Genes , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Glicina/metabolismo , Humanos , Camundongos , Neuroblastoma/genética , Serina/metabolismoRESUMO
HSCT is curative in SCD. Patients with HLA-identical sibling donor have an excellent outcome ranging from 90%-100% overall and event-free survival. However, due to the lack of matched sibling donors this option is out of reach for 70% of patients with SCD. The pool of potential donors needs to be extended. Transplantations from HLA-matched unrelated donors were reported to be less successful with shorter event-free survival and higher incidences of complications including graft-vs-host disease, especially in patients with advanced stage SCD. Here we report transplantation outcomes for 25 children with SCD transplanted using HLA-matched grafts from related or unrelated donors. Overall survival was 100% with no severe (grade III-IV) graft-vs-host disease and a 12% rejection rate. Mixed donor chimerisms only occurred in transplantations from siblings, while transplantations from unrelated donors resulted in either complete donor chimerism or rejection. Despite the small patient number, overall and disease-free survival for unrelated donor transplantations is excellent in this cohort. The advanced disease state, higher alloreactive effect and stronger immunosuppression in unrelated donor transplantations raises patient risk, for which possible solutions could be found in optimization of transplant preparation, graft manipulation or haploidentical transplantation using T cell receptor α/ß-depleted grafts.
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Anemia Falciforme/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Irmãos , Doadores não Relacionados , Adolescente , Anemia Falciforme/sangue , Anemia Falciforme/imunologia , Anemia Falciforme/mortalidade , Biomarcadores/sangue , Criança , Pré-Escolar , Quimerismo , Intervalo Livre de Doença , Feminino , Seguimentos , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/epidemiologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Antígenos HLA/imunologia , Teste de Histocompatibilidade , Humanos , Lactente , Isoanticorpos/sangue , Isoanticorpos/imunologia , Masculino , Estudos Retrospectivos , Transplante Homólogo/métodos , Resultado do Tratamento , Adulto JovemRESUMO
Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.
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Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Recombinação Genética/genética , Telomerase/genética , Telomerase/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Cromatina/genética , Cromatina/metabolismo , Cromossomos Humanos Par 5/genética , DNA Helicases/genética , Metilação de DNA , Elementos Facilitadores Genéticos/genética , Ativação Enzimática/genética , Amplificação de Genes/genética , Inativação Gênica , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/classificação , Neuroblastoma/enzimologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Prognóstico , RNA Mensageiro/análise , RNA Mensageiro/genética , Risco , Translocação Genética/genética , Regulação para Cima/genética , Proteína Nuclear Ligada ao XRESUMO
Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.
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Modelos Animais de Doenças , Neuroblastoma/genética , Neuroblastoma/patologia , Neoplasias Abdominais/genética , Neoplasias Abdominais/patologia , Animais , Feminino , Heterogeneidade Genética , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias Torácicas/genética , Neoplasias Torácicas/patologia , Células Tumorais CultivadasRESUMO
The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Humanos , Neuroblastoma/metabolismo , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
BACKGROUND: New strategies to optimize donor selection for hematopoietic stem cell transplantation (HSCT) have mainly been evaluated in adults, but the disease spectrum requiring HSCT differs significantly in children and has consequences for the risk of complications, such as graft-versus-host disease (GvHD). PROCEDURES: Here we evaluated whether HLA-DPB1 and Predicted Indirectly ReCognizable HLA-Epitope (PIRCHE) matching can improve donor selection and minimize risks specific for a pediatric cohort undergoing HSCT in Berlin between 2014 and 2016. RESULTS: The percentage of HLA-DPB1-mismatched HSCT in the pediatric cohort was in line with the general distribution among matched unrelated donor HSCT. Nonpermissive HLA-DPB1 mismatches were not associated with a higher incidence of GvHD, but the incidence of relapse was higher in patients undergoing HSCT from HLA-DPB1-matched transplantations. High PIRCHE-I scores were associated with a significantly higher risk for developing GvHD in patients undergoing HSCT from nine of ten matched unrelated donors. This finding persisted after including HLA-DPB1 into the PIRCHE analysis. CONCLUSIONS: Implementing PIRCHE typing in the donor selection process for HSCT in children could particularly benefit children with nonmalignant diseases and support further validation of PIRCHE-based donor selection in a larger number of children treated at different sites.
Assuntos
Seleção do Doador , Epitopos/imunologia , Doença Enxerto-Hospedeiro/mortalidade , Cadeias beta de HLA-DP/imunologia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/mortalidade , Recidiva Local de Neoplasia/terapia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/imunologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Teste de Histocompatibilidade , Humanos , Lactente , Masculino , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/mortalidade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Doadores não Relacionados , Adulto JovemRESUMO
Neuroblastoma is a common childhood cancer with almost a third of those affected still dying, thus new therapeutic strategies need to be explored. Current experimental therapies focus mostly on inhibiting oncogenic transcription factor signalling. Although LIN28B, DICER and other RNA-binding proteins (RBPs) have reported roles in neuroblastoma development and patient outcome, the role of RBPs in neuroblastoma is relatively unstudied. In order to elucidate novel RBPs involved in MYCN-amplified and other high-risk neuroblastoma subtypes, we performed differential mRNA expression analysis of RBPs in a large primary tumour cohort (n = 498). Additionally, we found via Kaplan-Meier scanning analysis that 685 of the 1483 tested RBPs have prognostic value in neuroblastoma. For the top putative oncogenic candidates, we analysed their expression in neuroblastoma cell lines, as well as summarised their characteristics and existence of chemical inhibitors. Moreover, to help explain their association with neuroblastoma subtypes, we reviewed candidate RBPs' potential as biomarkers, and their mechanistic roles in neuronal and cancer contexts. We found several highly significant RBPs including RPL22L1, RNASEH2A, PTRH2, MRPL11 and AFF2, which remain uncharacterised in neuroblastoma. Although not all RBPs appear suitable for drug design, or carry prognostic significance, we show that several RBPs have strong rationale for inhibition and mechanistic studies, representing an alternative, but nonetheless promising therapeutic strategy in neuroblastoma treatment.
Assuntos
Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Criança , Estudos de Coortes , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Oncogenes , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de RiscoRESUMO
BACKGROUND: High-risk neuroblastoma with N-Myc amplification remains a therapeutic challenge in paediatric oncology. Antagonism of pro-death Bcl-2 homology (BH) proteins to pro-survival BH members such as Mcl-1 and Bcl-2 has become a treatment approach, but previous studies suggest that a combined inhibition of Bcl-2 and Mcl-1 is necessary. TW-37 inhibits Mcl-1 and Bcl-2 with almost the same affinity. However, single-agent cytotoxicity of TW-37 in neuroblastoma cell lines has not been investigated. METHODS: Cell viability, apoptosis, proliferation and changes in growth properties were determined in SKNAS, IMR-5, SY5Y and Kelly cells after treatment with TW-37. After transfection with Mcl-1 or Bcl-2 siRNA, apoptosis and proliferation were investigated in Kelly cells. Mice with Kelly cell line xenografts were treated with TW-37 and tumor growth, survival and apoptosis were determined. RESULTS: Cell lines with N-Myc amplification were more sensitive to TW-37 treatment, IC50 values for IMR-5 and Kelly cells being 0.28 µM and 0.22 µM, compared to SY5Y cells and SKNAS cells (IC50 0.96 µM and 0.83 µM). Treatment with TW-37 resulted in increased apoptosis and reduced proliferation rates, especially in IMR5 and Kelly cells. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. TW-37 led to a decrease in tumor growth and a favorable survival (p = 0.0379) in a Kelly neuroblastoma xenografts mouse model. CONCLUSION: TW-37 has strong single-agent cytotoxicity in vitro and in vivo. Therefore, combined inhibition of Bcl-2/Mcl-1 by TW-37 in N-Myc amplified neuroblastoma may represent an interesting therapeutic strategy.
Assuntos
Benzamidas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Amplificação de Genes , Técnicas de Silenciamento de Genes , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonas/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. METHODS: CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. RESULTS: All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. CONCLUSION: Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Gangliosídeos/imunologia , Molécula L1 de Adesão de Célula Nervosa/imunologia , Receptores de Antígenos Quiméricos , Retinoblastoma/terapia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Citotoxicidade Imunológica , Feminino , Humanos , Lactente , Masculino , Retinoblastoma/imunologia , Retinoblastoma/metabolismo , Estudos Retrospectivos , Linfócitos T/imunologiaRESUMO
The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-ß signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-ß signaling cascade as well as direct inhibition of TGF-ß-responsive genes.
Assuntos
MicroRNAs/genética , Neuroblastoma/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Adesão Celular , Linhagem Celular , Proliferação de Células , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Neuroblastoma/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/genética , Transplante HeterólogoRESUMO
New drugs are crucially needed for children with cancer. The European Paediatric Regulation facilitates paediatric class waivers for drugs developed for diseases only occurring in adults. In this Review, we retrospectively searched oncology drugs that were class waivered between June, 2012, and June, 2015. 147 oncology class waivers were confirmed for 89 drugs. Mechanisms of action were then assessed as potential paediatric therapeutic targets by both a literature search and an expert review. 48 (54%) of the 89 class-waivered drugs had a mechanisms of action warranting paediatric development. Two (2%) class-waivered drugs were considered not relevant and 16 (18%) required further data. In light of these results, we propose five initiatives: an aggregated database of paediatric biological tumour drug targets; molecular profiling of all paediatric tumours at diagnosis and relapse; a joint academic-pharmaceutical industry preclinical platform to help analyse the activity of new drugs (Innovative Therapy for Children with Cancer Paediatric Preclinical Proof-of-Concept Platform); paediatric strategy forums; and the suppression of article 11b of the European Paediatric Regulation, which allows product-specific waivers on the grounds that the associated condition does not occur in children. These initiatives and a mechanism of action-based approach to drug development will accelerate the delivery of new therapeutic drugs for front-line therapy for those children who have unmet medical needs.