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PURPOSE: Outcomes for children with relapsed and refractory high-risk neuroblastoma (RR-HRNB) remain dismal. The BEACON Neuroblastoma trial (EudraCT 2012-000072-42) evaluated three backbone chemotherapy regimens and the addition of the antiangiogenic agent bevacizumab (B). MATERIALS AND METHODS: Patients age 1-21 years with RR-HRNB with adequate organ function and performance status were randomly assigned in a 3 × 2 factorial design to temozolomide (T), irinotecan-temozolomide (IT), or topotecan-temozolomide (TTo) with or without B. The primary end point was best overall response (complete or partial) rate (ORR) during the first six courses, by RECIST or International Neuroblastoma Response Criteria for patients with measurable or evaluable disease, respectively. Safety, progression-free survival (PFS), and overall survival (OS) time were secondary end points. RESULTS: One hundred sixty patients with RR-HRNB were included. For B random assignment (n = 160), the ORR was 26% (95% CI, 17 to 37) with B and 18% (95% CI, 10 to 28) without B (risk ratio [RR], 1.52 [95% CI, 0.83 to 2.77]; P = .17). Adjusted hazard ratio for PFS and OS were 0.89 (95% CI, 0.63 to 1.27) and 1.01 (95% CI, 0.70 to 1.45), respectively. For irinotecan ([I]; n = 121) and topotecan (n = 60) random assignments, RRs for ORR were 0.94 and 1.22, respectively. A potential interaction between I and B was identified. For patients in the bevacizumab-irinotecan-temozolomide (BIT) arm, the ORR was 23% (95% CI, 10 to 42), and the 1-year PFS estimate was 0.67 (95% CI, 0.47 to 0.80). CONCLUSION: The addition of B met protocol-defined success criteria for ORR and appeared to improve PFS. Within this phase II trial, BIT showed signals of antitumor activity with acceptable tolerability. Future trials will confirm these results in the chemoimmunotherapy era.
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Neuroblastoma , Topotecan , Niño , Humanos , Lactante , Preescolar , Adolescente , Adulto Joven , Adulto , Temozolomida/uso terapéutico , Irinotecán/uso terapéutico , Topotecan/efectos adversos , Bevacizumab/efectos adversos , Dacarbazina/efectos adversos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Neuroblastoma/patología , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversosRESUMEN
Outcomes for most patients with Ewing sarcoma (ES) have remained unchanged for the last 30 years, emphasising the need for more effective and tolerable treatments. We have hypothesised that using small-molecule inhibitors to kill the self-renewing chemotherapy-resistant cells (Ewing sarcoma cancer stem-like cells; ES-CSCs) responsible for progression and relapse could improve outcomes and minimise treatment-induced morbidities. For the first time, we demonstrate that ABCG1, a potential oncogene in some cancers, is highly expressed in ES-CSCs independently of CD133. Using functional models, transcriptomics and a bespoke in silico drug-repurposing pipeline, we have prioritised a group of tractable small-molecule inhibitors for further preclinical studies. Consistent with the cellular origin of ES, 21 candidate molecular targets of pluripotency, stemness and chemoresistance were identified. Small-molecule inhibitors to 13 of the 21 molecular targets (62%) were identified. POU5F1/OCT4 was the most promising new therapeutic target in Ewing sarcoma, interacting with 10 of the 21 prioritised molecular targets and meriting further study. The majority of small-molecule inhibitors (72%) target one of two drug efflux proteins, p-glycoprotein (n = 168) or MRP1 (n = 13). In summary, we have identified a novel cell surface marker of ES-CSCs and cancer/non-cancer drugs to targets expressed by these cells that are worthy of further preclinical evaluation. If effective in preclinical models, these drugs and drug combinations might be repurposed for clinical evaluation in patients with ES.
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BACKGROUND: Ewing sarcoma (ES) is the second most common primary bone malignancy, with an urgent need for new treatments. ES is associated with high rates of progression and relapse, driven by drug-resistant cells capable of migration, self-renewal and single-cell tumorigenesis, termed cancer stem-like cells (CSCs). Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteolytic enzyme, which, via direct and indirect mechanisms, digests four of the main types of collagen. This can be hijacked in malignancy for invasion and metastasis, with high expression predicting decreased survival in multiple cancers. In this study, we have examined the hypothesis that MT1-MMP is expressed by ES cells and explored the relationship between expression and outcomes. PROCEDURE: MT1-MMP expression in ES established cell lines, primary patient-derived cultures and daughter ES-CSCs was characterised by RNA sequencing, Western blotting, immunocytochemistry and flow cytometry. Immunohistochemistry was used to detect MT1-MMP in tumour biopsies, and the relationship between expression, event-free and overall survival examined. RESULTS: MT1-MMP was detected at both RNA and protein levels in five of six established cell lines, all primary cultures (n = 25) and all daughter ES-CSCs (n = 7). Immunohistochemistry of treatment-naïve biopsy tissue demonstrated that high MT1-MMP expression predicted decreased event-free and overall survival (p = .017 and .036, respectively; n = 47); this was not significant in multivariate analysis. CONCLUSIONS: MT1-MMP is expressed by ES cells, including ES-CSCs, making it a candidate therapeutic target. The level of MT1-MMP expression at diagnosis may be considered as a prognostic biomarker if validated by retrospective analysis of a larger cohort of clinical trial samples.
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Tumores Neuroectodérmicos Periféricos Primitivos , Sarcoma de Ewing , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Sarcoma de Ewing/tratamiento farmacológico , Estudios Retrospectivos , Recurrencia Local de Neoplasia , InmunohistoquímicaRESUMEN
PURPOSE: The development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets. METHODS: Thirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts. RESULTS: ES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation. CONCLUSIONS: Genes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.
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Proteínas Adaptadoras Transductoras de Señales/genética , Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Proteínas de Unión al Calcio/genética , Moléculas de Adhesión Celular Neuronal/genética , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/genética , Sarcoma de Ewing/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular Tumoral , Niño , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Pronóstico , Sarcoma de Ewing/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Células Tumorales Cultivadas , Vincristina/farmacologíaRESUMEN
Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.
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Biomarcadores de Tumor/sangre , Neoplasias Óseas/diagnóstico , ADN Tumoral Circulante/sangre , Sarcoma de Ewing/diagnóstico , Adolescente , Adulto , Biomarcadores de Tumor/genética , Neoplasias Óseas/sangre , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Estudios de Casos y Controles , Niño , Preescolar , ADN Tumoral Circulante/genética , Análisis Mutacional de ADN , Femenino , Humanos , Lactante , Biopsia Líquida/métodos , Masculino , Persona de Mediana Edad , Mutación , Sarcoma de Ewing/sangre , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Secuenciación Completa del Genoma , Adulto JovenRESUMEN
Background: Bone marrow involvement is an important aspect of determining staging of disease and treatment for childhood neuroblastoma. Current standard of care relies on microscopic examination of bone marrow trephine biopsies and aspirates respectively, to define involvement. Flow cytometric analysis of disaggregated tumour cells, when using a panel of neuroblastoma specific markers, allows for potentially less subjective determination of the presence of tumour cells. Methods: A retrospective review of sequential bone marrow trephine biopsies and aspirates, performed at Great Ormond Street Hospital, London, between the years 2015 and 2018, was performed to assess whether the addition of flow cytometric analysis to these standard of care methods provided concordant or additional information. Results: There was good concurrence between all three methods for negative results 216/302 (72%). Positive results had a concordance of 52/86 (61%), comparing samples positive by flow cytometry and positive by either or both cytology and histology. Of the remaining samples, 20/86 (23%) were positive by either or both cytology and histology, but negative by flow cytometry. Whereas 14/86 (16%) of samples were positive only by flow cytometry. Conclusions: Our review highlights the ongoing importance of expert cytological and histological assessment of bone marrow results. Flow cytometry is an objective, quantitative method to assess the level of bone marrow disease in aspirates. In this study, flow cytometry identified low-level residual disease that was not detected by cytology or histology. The clinical significance of this low-level disease warrants further investigation.
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Médula Ósea , Neuroblastoma , Biopsia/métodos , Médula Ósea/patología , Niño , Técnicas Citológicas , Citometría de Flujo/métodos , Humanos , Neuroblastoma/diagnóstico , Neuroblastoma/patologíaRESUMEN
PURPOSE: Circulating tumor cells (CTCs) serve as noninvasive tumor biomarkers in many types of cancer. Our aim was to detect CTCs from patients with neuroblastoma for use as predictive and pharmacodynamic biomarkers. EXPERIMENTAL DESIGN: We collected matched blood and bone marrow samples from 40 patients with neuroblastoma to detect GD2 +/CD45- neuroblastoma CTCs from blood and disseminated tumor cells (DTCs) from bone marrow using the Imagestream Imaging flow cytometer (ISx). In six cases, circulating free DNA (cfDNA) extracted from plasma isolated from the CTC sample was analyzed by high-density single-nucleotide polymorphism (SNP) arrays. RESULTS: CTCs were detected in 26 of 42 blood samples (1-264/mL) and DTCs in 25 of 35 bone marrow samples (57-291,544/mL). Higher numbers of CTCs in patients with newly diagnosed, high-risk neuroblastoma correlated with failure to obtain a complete bone marrow (BM) metastatic response after induction chemotherapy (P < 0.01). Ex vivo Nutlin-3 (MDM2 inhibitor) treatment of blood and BM increased p53 and p21 expression in CTCs and DTCs compared with DMSO controls. In five of six cases, cfDNA analyzed by SNP arrays revealed copy number abnormalities associated with neuroblastoma. CONCLUSIONS: This is the first study to show that CTCs and DTCs are detectable in neuroblastoma using the ISx, with concurrently extracted cfDNA used for copy number profiling, and may be useful as pharmacodynamic biomarkers in early-phase clinical trials. Further investigation is required to determine whether CTC numbers are predictive biomarkers of BM response to first-line induction chemotherapy.
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Biomarcadores de Tumor/sangre , Médula Ósea/patología , Citometría de Flujo/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imidazoles/farmacología , Células Neoplásicas Circulantes/patología , Neuroblastoma/patología , Piperazinas/farmacología , Biomarcadores de Tumor/genética , Médula Ósea/efectos de los fármacos , Variaciones en el Número de Copia de ADN , Humanos , Células Neoplásicas Circulantes/efectos de los fármacos , Neuroblastoma/sangre , Neuroblastoma/tratamiento farmacológico , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidoresRESUMEN
Despite intensive treatment, 50% of children with high-risk neuroblastoma (HR-NB) succumb to their disease. Progression through current trials evaluating the efficacy of new treatments for children with HR disease usually depends on an inadequate response to induction chemotherapy, assessed using imaging modalities. In this study, we sought to identify circulating biomarkers that might be detected in a simple blood sample to predict patient response to induction chemotherapy. Since exosomes released by tumor cells can drive tumor growth and chemoresistance, we tested the hypothesis that exosomal microRNA (exo-miRNAs) in blood might predict response to induction chemotherapy. The exo-miRNAs expression profile in plasma samples collected from children treated in HR-NBL-1/SIOPEN before and after induction chemotherapy was compared to identify a three exo-miRs signature that could discriminate between poor and good responders. Exo-miRNAs expression also provided a chemoresistance index predicting the good or poor prognosis of HR-NB patients.
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Patients with osteosarcoma (OST) frequently relapse with drug resistant disease, consistent with the hypothesis that tumours contain a cancer stem-like cell (CSCs) population that survives chemotherapy to re-populate the tumour at local or metastatic sites. We describe a dual functional approach to isolate OST-CSCs and identify the ABC transporter proteins driving this population to reveal potential targets for the development of new treatments. OST-CSCs were isolated by selection in doxorubicin (OST-EC50â¯cells) and based on the ability to produce progeny from a single cell (HOS-EC50.SR cells). Pgp expression was increased in OST-EC50â¯cells, inducing resistance to doxorubicin, etoposide, vincristine and actinomycin D (pâ¯<â¯0.05). Increased expression of ABCG1 and Pgp protein in the HOS-EC50.SR cells induced resistance to etoposide and doxorubicin (pâ¯<â¯0.01), which was directly correlated with ABCG1 expression (râ¯>â¯0.88, pâ¯<â¯0.001). Pgp expression is increased in both the HOS-EC50â¯cells where it mediates MDR and the HOS-EC50.SR populations, whereas ABCG1 was only upregulated in the self-renewing drug resistant HOS-EC50.SR cells. Targeting ABCG1 and Pgp may eradicate the drug resistant self-renewing OST-CSCs, leading to improved outcomes for patients with OST.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Dactinomicina/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Etopósido/farmacología , Células HEK293 , Células Hep G2 , Humanos , Células MCF-7 , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Osteosarcoma/genética , Osteosarcoma/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vincristina/farmacologíaRESUMEN
Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1ß and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1ß and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1ß and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.
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Arginina/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Neuroblastoma/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Arginasa/metabolismo , Línea Celular Tumoral , Humanos , Interleucina-1beta/inmunología , Sistema de Señalización de MAP Quinasas , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Transgénicos , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células Mieloides/patología , Neuroblastoma/inmunología , Neuroblastoma/patología , Sarcoma de Ewing/inmunología , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
AIMS: The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. BACKGROUND: Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. METHODS: qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. RESULTS: The wild type α+/ß+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/ß- hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (pâ¯=â¯0.05) and primary cultures (pâ¯=â¯0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/ß+ hTERT (pâ¯=â¯0.04), and a strong positive association between MCPH1/BRIT1 and both α-/ß+ hTERT and α-/ß- hTERT (both pâ¯=â¯0.02). A positive association was observed between BRCA1 and α-/ß+ hTERT and α-/ß- hTERT expression (pâ¯=â¯0.003 and pâ¯=â¯0.04, respectively). CONCLUSIONS: These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/ß+).
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Proteína BRCA1/genética , Neoplasias Glandulares y Epiteliales/genética , Proteínas del Tejido Nervioso/genética , Neoplasias Ováricas/genética , Telomerasa/genética , Proteína BRCA1/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Epitelial de Ovario , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteínas del Citoesqueleto , Femenino , Expresión Génica , Genes Supresores de Tumor , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Estimación de Kaplan-Meier , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Glandulares y Epiteliales/mortalidad , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/mortalidad , Telomerasa/metabolismo , TranscriptomaRESUMEN
BACKGROUND: The purpose of this study was to evaluate whether levels of neuroblastoma mRNAs in bone marrow and peripheral blood from stage M infants (≤12 months of age at diagnosis, MYCN amplified) and toddlers (between 12 and 18 months, any MYCN status) predict event-free survival (EFS). METHODS: Bone marrow aspirates and peripheral blood samples from 97 infants/toddlers enrolled in the European High-Risk Neuroblastoma trial were collected at diagnosis in PAXgene™ blood RNA tubes. Samples were analyzed by reverse transcription quantitative polymerase chain reaction according to standardized procedures. RESULTS: Bone marrow tyrosine hydroxylase (TH) or paired-like homeobox 2b (PHOX2B) levels in the highest tertile were associated with worse EFS; hazard ratios, adjusted for age and MYCN status, were 1.5 and 1.8 respectively. Expression of both TH and PHOX2B in the highest tertile predicted worse outcome (p = 0.015), and identified 20 (23%) infants/toddlers with 5-year EFS of 20% (95%CI: 4%-44%). Prognostic significance was maintained after adjusting for over-fitting bias (p = 0.038), age and MYCN status. In peripheral blood, PHOX2B levels in the highest tertile predicted a two-fold increased risk of an event (p = 0.032), and identified 23 (34%) infants/toddlers with 5-year EFS of 29% (95%CI: 12%-48%). Time-dependent receiver operating characteristic analysis confirmed the prognostic value of combined TH and PHOX2B in bone marrow and of PHOX2B in peripheral blood during the first year of follow-up. CONCLUSIONS: High levels of bone marrow TH and PHOX2B and of peripheral blood PHOX2B at diagnosis allow early identification of a group of high-risk infant and toddlers with neuroblastoma who may be candidates for alternative treatments. Integration with additional biomarkers, as well as validation in additional international trials is warranted.
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Biomarcadores de Tumor/análisis , Proteínas de Homeodominio/análisis , Neuroblastoma/mortalidad , Factores de Transcripción/análisis , Tirosina 3-Monooxigenasa/análisis , Área Bajo la Curva , Femenino , Proteínas de Homeodominio/biosíntesis , Humanos , Lactante , Recién Nacido , Estimación de Kaplan-Meier , Masculino , Neuroblastoma/metabolismo , Pronóstico , Supervivencia sin Progresión , Modelos de Riesgos Proporcionales , ARN Mensajero/análisis , Curva ROC , Sensibilidad y Especificidad , Factores de Transcripción/biosíntesis , Tirosina 3-Monooxigenasa/biosíntesisRESUMEN
Purpose More than two decades ago, an international working group established the International Neuroblastoma Response Criteria (INRC) to assess treatment response in children with neuroblastoma. However, this system requires modification to incorporate modern imaging techniques and new methods for quantifying bone marrow disease that were not previously widely available. The National Cancer Institute sponsored a clinical trials planning meeting in 2012 to update and refine response criteria for patients with neuroblastoma. Methods Multidisciplinary investigators from 13 countries reviewed data from published trials performed through cooperative groups, consortia, and single institutions. Data from both prospective and retrospective trials were used to refine the INRC. Monthly international conference calls were held from 2011 to 2015, and consensus was reached through review by working group leadership and the National Cancer Institute Clinical Trials Planning Meeting leadership council. Results Overall response in the revised INRC will integrate tumor response in the primary tumor, soft tissue and bone metastases, and bone marrow. Primary and metastatic soft tissue sites will be assessed using Response Evaluation Criteria in Solid Tumors (RECIST) and iodine-123 (123I) -metaiodobenzylguanidine (MIBG) scans or [18F]fluorodeoxyglucose-positron emission tomography scans if the tumor is MIBG nonavid. 123I-MIBG scans, or [18F]fluorodeoxyglucose-positron emission tomography scans for MIBG-nonavid disease, replace technetium-99m diphosphonate bone scintigraphy for osteomedullary metastasis assessment. Bone marrow will be assessed by histology or immunohistochemistry and cytology or immunocytology. Bone marrow with ≤ 5% tumor involvement will be classified as minimal disease. Urinary catecholamine levels will not be included in response assessment. Overall response will be defined as complete response, partial response, minor response, stable disease, or progressive disease. Conclusion These revised criteria will provide a uniform assessment of disease response, improve the interpretability of clinical trial results, and facilitate collaborative trial designs.
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Neoplasias de la Médula Ósea/diagnóstico , Neoplasias de la Médula Ósea/patología , Neoplasias Óseas/diagnóstico por imagen , Neuroblastoma/diagnóstico por imagen , Tomografía de Emisión de Positrones , Neoplasias de los Tejidos Blandos/diagnóstico por imagen , 3-Yodobencilguanidina , Neoplasias de la Médula Ósea/química , Neoplasias de la Médula Ósea/secundario , Neoplasias Óseas/secundario , Consenso , Fluorodesoxiglucosa F18 , Humanos , Inmunohistoquímica , Neuroblastoma/secundario , Neuroblastoma/terapia , Radiofármacos , Criterios de Evaluación de Respuesta en Tumores Sólidos , Neoplasias de los Tejidos Blandos/secundarioRESUMEN
BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society.
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Enfermedades de la Médula Ósea/diagnóstico , Enfermedades de la Médula Ósea/etiología , Médula Ósea/patología , Neuroblastoma/patología , Biopsia/métodos , Examen de la Médula Ósea/métodos , Humanos , Inmunohistoquímica , Invasividad Neoplásica , Metástasis de la Neoplasia , Neuroblastoma/terapia , Reacción en Cadena de la PolimerasaRESUMEN
Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90ß decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90ß, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90ß.
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Regulación de la Expresión Génica/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transporte de Proteínas/fisiología , Antineoplásicos/farmacología , Línea Celular Tumoral , Dactinomicina/farmacología , Doxorrubicina/farmacología , Fenretinida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
PURPOSE: To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk. METHODS: RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy. RESULTS: High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. CONCLUSION: High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.
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Biomarcadores de Tumor/metabolismo , Médula Ósea/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , ARN Mensajero/sangre , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/sangre , Carboplatino/administración & dosificación , Niño , Preescolar , Cisplatino/administración & dosificación , Simulación por Computador , Ciclofosfamida/administración & dosificación , Supervivencia sin Enfermedad , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Etopósido/administración & dosificación , Proteínas de Homeodominio/genética , Humanos , Quimioterapia de Inducción , Lactante , Estimación de Kaplan-Meier , Proteínas Asociadas a Microtúbulos/genética , Neuroblastoma/tratamiento farmacológico , Neuropéptidos/genética , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Factores de Transcripción/genética , Tirosina 3-Monooxigenasa/genética , Vincristina/administración & dosificaciónRESUMEN
In many cancers, including neuroblastoma, microRNA (miRNA) expression profiling of peripheral blood (PB) and bone marrow (BM) may increase understanding of the metastatic process and lead to the identification of clinically informative biomarkers. The quality of miRNAs in PB and BM samples archived in PAXgene™ blood RNA tubes from large-scale clinical studies and the identity of reference miRNAs for standard reporting of data are to date unknown. In this study, we evaluated the reliability of expression profiling of 377 miRNAs using quantitative polymerase chain reaction (qPCR) in PB and BM samples (n=90) stored at -80 °C for up to 5 years in PAXgene™ blood RNA tubes. There was no correlation with storage time and variation of expression for any single miRNA (r < 0.50). The profile of miRNAs isolated as small RNAs or co-isolated with small/large RNAs was highly correlated (r=0.96). The mean expression of all miRNAs and the geNorm program identified miR-26a, miR-28-5p, and miR-24 as the most stable reference miRNAs. This study describes detailed methodologies for reliable miRNA isolation and profiling of PB and BM, including reference miRNAs for qPCR normalization, and demonstrates the suitability of clinical samples archived at -80 °C into PAXgene™ blood RNA tubes for miRNA expression studies.
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Médula Ósea/metabolismo , Perfilación de la Expresión Génica , MicroARNs/sangre , MicroARNs/genética , Niño , Hematopoyesis , Humanos , MicroARNs/aislamiento & purificación , Reacción en Cadena de la PolimerasaRESUMEN
Glutathione (GSH) is implicated in drug resistance mechanisms of several cancers and is a key regulator of cell death pathways within cells. We studied Ewing's sarcoma family of tumours (ESFT) cell lines and three mechanistically distinct anticancer agents (fenretinide, doxorubicin, and vincristine) to investigate whether the GSH antioxidant system is involved in the reduced sensitivity to these chemotherapeutic agents in hypoxia. Cell viability and death were assessed by the trypan blue exclusion assay and annexin V-PI staining, respectively. Hypoxia significantly decreased the sensitivity of all ESFT cell lines to fenretinide-induced death, whereas the effect of doxorubicin or vincristine was marginal and cell-line-specific. The response of the GSH antioxidant system in ESFT cell lines to hypoxia was variable and also cell-line-specific, although the level of GSH appeared to be most dependent on de novo biosynthesis rather than recycling. RNAi-mediated knockdown of key GSH regulatory enzymes γ-glutamylcysteine synthetase or glutathione disulfide reductase partially reversed the hypoxia-induced resistance to fenretinide, and increasing GSH levels using N-acetylcysteine augmented the hypoxia-induced resistance in a cell line-specific manner. These observations are consistent with the conclusion that the role of the GSH antioxidant system in modulating the sensitivity of ESFT cells to fenretinide is heterogeneous depending on environment and cell type. This is likely to limit the value of targeting GSH as a therapeutic strategy to overcome hypoxia-induced drug resistance in ESFT. Whether targeting the GSH antioxidant system in conjunction with other therapeutics may benefit some patients with ESFT remains to be seen.
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Antioxidantes/metabolismo , Fenretinida/uso terapéutico , Glutatión/metabolismo , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/patología , Acetilcisteína/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Recuento de Células , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Suplementos Dietéticos , Ensayos de Selección de Medicamentos Antitumorales , Fenretinida/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sarcoma de Ewing/enzimologíaRESUMEN
Reactive oxygen species (ROS) are highly reactive by-products of energy production that can have detrimental as well as beneficial effects. Unchecked, high levels of ROS result in an imbalance of cellular redox state and oxidative stress. High levels of ROS have been detected in most cancers, where they promote tumor development and progression. Many anticancer agents work by further increasing cellular levels of ROS, to overcome the antioxidant detoxification capacity of the cancer cell and induce cell death. However, adaptation of the level of cellular antioxidants can lead to drug resistance. The challenge for the design of effective cancer therapeutics exploiting oxidative stress is to tip the cellular redox balance to induce ROS-dependent cell death but without increasing the antioxidant activity of the cancer cell or inducing toxicity in normal cells.