ABSTRACT
BACKGROUND: Long-term survival of high-risk neuroblastoma patients is still below 50% despite intensive multimodal treatment. This trial aimed to address whether the addition of two topotecan-containing chemotherapy courses compared to standard induction therapy improves event-free survival (EFS) of these patients. PATIENTS AND METHODS: An open-label, multicenter, prospective randomized controlled trial was carried out at 58 hospitals in Germany and Switzerland. Patients aged 1-21 years with stage 4 neuroblastoma and patients aged 6 months to 21 years with MYCN-amplified tumors were eligible. The primary endpoint was EFS. Patients were randomly assigned to standard induction therapy with six chemotherapy courses or to experimental induction chemotherapy starting with two additional courses of topotecan, cyclophosphamide, and etoposide followed by standard induction chemotherapy (eight courses in total). After induction chemotherapy, all patients received high-dose chemotherapy with autologous hematopoietic stem cell rescue and isotretinoin for consolidation. Radiotherapy was applied to patients with active tumors at the end of induction chemotherapy. RESULTS: Of 536 patients enrolled in the trial, 422 were randomly assigned to the control arm (n = 211) and the experimental arm (n = 211); the median follow-up time was 3.32 years (interquartile range 1.65-5.92). At data lock, the 3-year EFS of experimental and control patients was 34% and 32% [95% confidence Interval (CI) 28% to 40% and 26% to 38%; P = 0.258], respectively. Similarly, the 3-year overall survival of the patients did not differ [54% and 48% (95% CI 46% to 62% and 40% to 56%), respectively; P = 0.558]. The response to induction chemotherapy was not different between the arms. The median number of non-fatal toxicities per patient was higher in the experimental group while the median number of toxicities per chemotherapy course was not different. CONCLUSION: While the burden for the patients was increased by prolonging the induction chemotherapy and the toxicity, the addition of two topotecan-containing chemotherapy courses did not improve the EFS of high-risk neuroblastoma patients and thus cannot be recommended. CLINICAL TRIALS. GOV NUMBER: NCT number 03042429.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Induction Chemotherapy , Neuroblastoma , Adolescent , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Child , Child, Preschool , Disease-Free Survival , Germany , Humans , Infant , Neuroblastoma/drug therapy , Prospective Studies , Switzerland , Treatment Outcome , Young AdultABSTRACT
PURPOSE: In 2014, we published the qPET method to quantify fluorodeoxyglucose positron emission tomography (FDG-PET) responses. Analysis of the distribution of the quantified signals suggested that a clearly abnormal FDG-PET response corresponds to a visual Deauville score (vDS) of 5 and high qPET values ≥ 2. Evaluation in long-term outcome data is still pending. Therefore, we analyzed progression-free survival (PFS) by early FDG-PET response in a subset of the GPOH-HD2002 trial for pediatric Hodgkin lymphoma (PHL). PATIENTS/METHODS: Pairwise FDG-PET scans for initial staging and early response assessment after two cycles of chemotherapy were available in 93 PHL patients. vDS and qPET measurement were performed and related to PFS. RESULTS: Patients with a qPET value ≥ 2.0 or vDS of 5 had 5-year PFS rates of 44%, respectively 50%. Those with qPET values < 2.0 or vDS 1 to 4 had 5-year PFS rates of 90%, respectively 80%. The positive predictive value of FDG-PET response assessment increased from 18% (9%; 33%) using a qPET threshold of 0.95 (vDS ≤ 3) to 30% (13%; 54%) for a qPET threshold of 1.3 (vDS ≤ 4) and to 56% (23%; 85%) when the qPET threshold was ≥ 2.0 (vDS 5). The negative predictive values remained stable at ≥92% (CI: 82%; 98%). CONCLUSION: Only strongly enhanced residual FDG uptake in early response PET (vDS 5 or qPET ≥ 2, respectively) seems to be markedly prognostic in PHL when treatment according to the GPOH-HD-2002 protocol is given.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Fluorodeoxyglucose F18/metabolism , Hodgkin Disease/pathology , Positron-Emission Tomography/methods , Radiopharmaceuticals/metabolism , Child , Clinical Trials as Topic , Female , Follow-Up Studies , Hodgkin Disease/diagnostic imaging , Hodgkin Disease/drug therapy , Hodgkin Disease/metabolism , Humans , Male , Prognosis , ROC Curve , Survival RateABSTRACT
BACKGROUND: The hydrophobic triterpenes, oleanolic and betulinic acid as well as the hydrophilic mistletoe lectins and viscotoxins possess anticancer properties. They do all occur in combination in European mistletoe (Viscum album L.). Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We have previously shown that mistletoe lectins and triterpene acids are effective against Ewing sarcoma in vitro, ex vivo and in vivo. METHODS: We recreated a total mistletoe effect (viscumTT) by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilised with cyclodextrins and analysed the effects of viscumTT and the single extracts on TC-71 Ewing sarcoma cells in vitro by transcriptomic and proteomic profiling. RESULTS: Treatment with the extracts strongly impacted Ewing sarcoma cell gene and protein expression. Apoptosis-associated and stress-activated genes were upregulated, proteasomal protein abundance enhanced and ribosomal and spliceosomal proteins downregulated. The mechanism of action of viscum, TT and viscumTT in TC-71 and MHH-ES-1 cells suggests the involvement of the unfolded protein response. While viscum and viscumTT extract treatment indicate response to oxidative stress and activation of stress-mediated MAPK signalling, TT extract treatment suggests the involvement of TLR signalling and autophagy. CONCLUSIONS: Since the combinatory extract viscumTT exerts highly effective pro-apoptotic effects on Ewing sarcoma cells in vitro, this phytopolychemotherapy could be a promising adjuvant therapeutic option for paediatric patients with Ewing sarcoma.
Subject(s)
Oxidative Stress/drug effects , Plant Extracts/pharmacology , Plant Lectins/pharmacology , Plant Proteins/pharmacology , Sarcoma, Ewing/metabolism , Triterpenes/pharmacology , Viscum album/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Apoptosis , Autophagy , Cell Proliferation , Humans , Mitogen-Activated Protein Kinases/metabolism , Neoplasm Proteins/metabolism , Oleanolic Acid/pharmacology , Oleanolic Acid/therapeutic use , Pentacyclic Triterpenes , Phytotherapy , Plant Extracts/therapeutic use , Plant Lectins/therapeutic use , Plant Proteins/therapeutic use , Proteasome Endopeptidase Complex/metabolism , Proteome , Proteomics , Sarcoma, Ewing/drug therapy , Signal Transduction , Transcriptome , Triterpenes/therapeutic use , Tumor Cells, Cultured , Betulinic AcidABSTRACT
HYPOTHESIS: Understanding the coarsening behavior of foams is of great interest for their deliberate design. In order to systematically quantify the influence of surfactants and other chemical parameters, identifying robust descriptive features of observed foam aging dynamics is essential. Existing coarsening theories for both wet and dry foams provide concise models with respective descriptive parameters. EXPERIMENT: Multiple micro computed tomography scans of moderately wet polydisperse ß-Lactoglobulin foam are recorded over a period of 15min. The growth behavior of a large fraction of about 5×10(4) pores that constitute the imaged volume is individually observed and statistically analyzed as a function of pore radius as well as number of neighboring pores. FINDINGS: The three-dimensional analog of von Neumann's law for dry foams by Glazier is confirmed as a suiting empirical model, whereby a critical number of 13±7 neighbors and a diffusion coefficient of (1.8±0.8)×10(-11)m(2)/s are found for an exemplary sample. The pores growth can as well be related to their radius by means of Lemlich's coarsening model for wet foams though, whereby a critical radius marking the transition between shrinkage and growths is found to be Rc=(300±85)µm. Although different, both models fit similarly well given the broad variance of the observed growth rates.
Subject(s)
Lactoglobulins/chemistry , Lactoglobulins/isolation & purification , Particle Size , Surface PropertiesABSTRACT
Chronic stress has been associated with several negative health outcomes and psychopathological conditions. One source of chronic stress might be from ones social environment (e.g., being excluded from a group, losing a loved one, etc.). Specifically, social instability, or frequent changes in the social environment, can result in both physiological and behavioral stress responses. Corticosterone is the primary stress-responsive biomarker in rodents, and it reflects the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Historically, research on the effects of stress has relied on collection of blood, saliva, or other bodily fluids that yield information about moment-to-moment changes in stress physiology. Recently, new sampling techniques involving quantification of glucocorticoids in hair has allowed researchers to view patterns of stress physiology over extended periods of time. This study assessed the effects of chronic social instability on corticosterone levels in female mice. Mice that were subjected to social instability showed elevated hair corticosterone compared to baseline levels and as compared to controls. Additionally, when mice were returned to stable social environments, corticosterone levels returned to levels comparable to baseline and to control animals. This suggests that the corticosterone in hair from female mice can serve as a useful biomarker of chronic stress, and that social instability is a sufficient stressor to elicit an extended HPA response.
Subject(s)
Anxiety/metabolism , Behavior, Animal/physiology , Corticosterone/metabolism , Hair/metabolism , Social Environment , Stress, Psychological/metabolism , Animals , Disease Models, Animal , Female , MiceABSTRACT
Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine ß-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.
Subject(s)
Drug Evaluation, Preclinical/methods , Integrases/genetics , Neuroblastoma/pathology , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Promoter Regions, Genetic , Transgenes , Animals , Disease Models, Animal , Female , Gene Expression Profiling , Humans , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Microarray Analysis , N-Myc Proto-Oncogene Protein , Neuroblastoma/genetics , Tumor Cells, CulturedSubject(s)
Congresses as Topic , Medical Oncology , Pediatrics , Periodicals as Topic , Publishing , Child , Germany , HumansABSTRACT
Treatment for neuroblastoma, the most common extracranial childhood tumor, spans a broad range of aggressiveness that mirrors the risk profiles of disease subtypes, with high-risk neuroblastoma still presenting a clinical challenge. Currently, most patients with relapsed neuro-blastoma die of disease and present a major challenge for treatment. New therapeutic options are urgently needed to improve patient survival. Activating mutations in the gene encoding the anaplastic lymphoma kinase (ALK) remain the most frequent druggable mutations identified in neuroblastomas to date. Preclinical data support an oncogene addiction of neuroblastoma cells to mutated ALK and demonstrate that ALK inhibitory therapy strongly combats tumor models. Most recently, pediatric phase I testing has been completed for the first approved ALK inhibitor, Crizotinib, showing very encouraging antitumoral results in neuroblastoma patients. Subsequently, an international phase I study with the second generation ALK inhibitor, LDK-378, will be launched that makes ALK inhibitory therapy also available to pediatric patients in Germany.
Subject(s)
Drug Delivery Systems , Neuroblastoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Anaplastic Lymphoma Kinase , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Child , Clinical Trials, Phase I as Topic , Crizotinib , DNA Mutational Analysis , Drug Approval , Germany , Humans , Neoplasm Staging , Neuroblastoma/genetics , Neuroblastoma/pathology , Protein Kinase Inhibitors/adverse effects , Pyrazoles/adverse effects , Pyrazoles/therapeutic use , Pyridines/adverse effects , Pyridines/therapeutic use , Pyrimidines/adverse effects , Pyrimidines/therapeutic use , Receptor Protein-Tyrosine Kinases/genetics , Sulfones/adverse effects , Sulfones/therapeutic useSubject(s)
Granulosa Cell Tumor/diagnosis , Hypercalcemia/diagnosis , Paraneoplastic Syndromes/diagnosis , Biomarkers, Tumor/blood , Diagnosis, Differential , Female , Granulosa Cell Tumor/pathology , Granulosa Cell Tumor/surgery , Humans , Hypercalcemia/pathology , Hypercalcemia/surgery , Magnetic Resonance Imaging , Neoplasm Staging , Ovariectomy , Ovary/pathology , Paraneoplastic Syndromes/pathology , Paraneoplastic Syndromes/surgery , Renal Insufficiency/diagnosisABSTRACT
There are approximately 40 new cases of retinoblastoma in Germany per year. Children in whom the tumour is detected when still intraocular have an excellent overall survival rate (> 95%). However, the prognosis of metastasised retinoblastoma remains poor. About 40% of retinoblastoma patients have tumours in both eyes. For these children in particular it is important to save the eye and visual function as much as possible. There are several options for conservative treatment of localised retinoblastoma including laser coagulation, thermotherapy, cryotherapy, brachytherapy and chemotherapy. In recent years, systemic chemotherapy has become the established standard for primary treatment of intraocular retinoblastoma. In case series, intra-arterial, intravitreal and periocular applications of chemotherapy were also shown to be effective in treating intraocular retinoblastoma. Genetic testing is an integral part of the routine diagnostics of all patients. Mutation analysis of tumour material is invaluable for identification of somatic mutations including mutational mosaicism. Genetic testing also identifies children with heritable retinoblastoma, which represent 50% of cases. These children also have a predisposition for the development of tumours outside of the eye (second primary neoplasm). To adequately address these and other late effects in survivors of retinoblastoma, a multidisciplinary approach is needed that optimises therapy and long-term follow-up. Upcoming multicentre clinical trials will evaluate treatment concepts for localised and metastasised retinoblastoma to improve survival rates and quality of life of children with retinoblastoma. This article was translated and modified and was primarily published in Klin Padiatr 2012; 224: 339-347.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Eye Enucleation , Postoperative Complications/prevention & control , Retinal Neoplasms/diagnosis , Retinal Neoplasms/therapy , Retinoblastoma/diagnosis , Retinoblastoma/therapy , Visual Acuity/drug effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Chemoradiotherapy, Adjuvant , Child , Disease Progression , Drug Administration Routes , Genetic Predisposition to Disease , Humans , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , Retinal Neoplasms/genetics , Retinal Neoplasms/pathology , Retinoblastoma/genetics , Retinoblastoma/pathology , Treatment OutcomeABSTRACT
Neuroblastoma is an embryonal tumor with a heterogeneous clinical course. The tumor is presumed to be derived from the neural crest, but the cells of origin remain to be determined. To date, few recurrent genetic changes contributing to neuroblastoma formation, such as amplification of the MYCN oncogene and activating mutations of the ALK oncogene, have been identified. The possibility to model neuroblastoma in mice allows investigation of the cell of origin hypothesis in further detail. Here we present the evidence that murine neural crest progenitor cells can give rise to neuroblastoma upon transformation with MYCN or ALK(F1174L). For this purpose we used JoMa1, a multipotent neural crest progenitor cell line, which is kept in a viable and undifferentiated state by a tamoxifen-activated c-Myc transgene (c-MycER(T)). Expression of MYCN or ALK(F1174L), one of the oncogenic ALK variants identified in primary neuroblastomas, enabled these cells to grow independently of c-MycER(T) activity in vitro and caused formation of neuroblastoma-like tumors in vivo in contrast to parental JoMa1 cells and JoMa1 cells-expressing TrkA or GFP. Tumorigenicity was enhanced upon serial transplantation of tumor-derived cells, and tumor cells remained susceptible to the MYC-inhibitor, NBT-272, indicating that cell growth depended on functional MYCN. Our findings support neural crest progenitor cells as the precursor cells of neuroblastoma, and indicate that neuroblastomas arise as their malignant progeny.
Subject(s)
Neoplastic Stem Cells/pathology , Neural Crest/pathology , Neuroblastoma/genetics , Neuroblastoma/pathology , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Stem Cells/pathology , Anaplastic Lymphoma Kinase , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Humans , Mice , Mice, Nude , Mice, Transgenic , N-Myc Proto-Oncogene Protein , Neoplastic Stem Cells/metabolism , Neural Crest/metabolism , Neuroblastoma/metabolism , Nuclear Proteins/biosynthesis , Nuclear Proteins/metabolism , Oncogene Proteins/biosynthesis , Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/metabolism , Stem Cells/metabolism , Transfection , Transplantation, HeterologousABSTRACT
MicroRNAs (miRNAs) contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs was associated with poor patient survival when underexpressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are overexpressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic overexpression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is upregulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3'-untranslated region, explaining the mechanism by which SOX2 is downregulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340-mediated downregulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis.
Subject(s)
Epigenesis, Genetic/genetics , Gene Regulatory Networks/genetics , MicroRNAs/genetics , Neuroblastoma/etiology , Neuroblastoma/genetics , 3' Untranslated Regions/genetics , Cell Line, Tumor , Computational Biology , DNA Methylation/drug effects , DNA Methylation/genetics , Epigenesis, Genetic/drug effects , Gene Regulatory Networks/drug effects , Genomics , Humans , Neuroblastoma/pathology , SOXB1 Transcription Factors/genetics , Survival Analysis , Tretinoin/pharmacologySubject(s)
Antineoplastic Agents/therapeutic use , Evidence-Based Medicine/legislation & jurisprudence , Guideline Adherence/legislation & jurisprudence , Hematologic Neoplasms/drug therapy , National Health Programs/legislation & jurisprudence , Neoplasms/drug therapy , Adolescent , Child , Combined Modality Therapy , Cooperative Behavior , Critical Care/legislation & jurisprudence , Disease Progression , Germany , Hematologic Neoplasms/diagnosis , Hematologic Neoplasms/pathology , Humans , Interdisciplinary Communication , International Cooperation , Neoplasm Staging , Neoplasms/diagnosis , Neoplasms/pathology , Registries , Societies, Medical , Treatment OutcomeABSTRACT
Retinoblastoma affects approximately 40 children in Germany per year. Most children are diagnosed early with localized intraocular disease, and the overall survival rate exceeds 95%. However, the prognosis of metastasized retinoblastoma remains poor. In 40% of the patients, retinoblastoma occurs bilaterally and, especially for these children, the salvage of the eye and visual function is of major importance. The variety of conservative treatment options for localized retinoblastoma includes laser coagulation, thermotherapy, cryotherapy, brachytherapy and chemotherapy. While systemic chemotherapy has nearly completely replaced external beam radiotherapy in the primary treatment of intraocular retinoblastoma, intra-arterial, intravitreal and periocular application of chemotherapy was also shown to be effective in treating intraocular retinoblastoma in case series. Genetic testing is an integral part of the routine diagnostics of all patients. Available tumor material should be analyzed to detect mutational mosaicism, that affects >10% of children with unilateral retinoblastoma. Genetic testing also identifies children with heritable (50% of patients) retinoblastoma. These children have a genetic predisposition for second malignancies. For this reason, late effects are an increasing concern and the care of patients with retinoblastoma requires a multidisciplinary approach to tailor therapy and long-term follow-up. Multicenter clinical trials are being developed to evaluate evidence-based treatment concepts for localized and metastasized retinoblastoma to improve survival rates and quality of life of children with retinoblastoma.
Subject(s)
Retinal Neoplasms/diagnosis , Retinal Neoplasms/therapy , Retinoblastoma/diagnosis , Retinoblastoma/therapy , Vision Disorders/diagnosis , Vision Disorders/prevention & control , Vision Disorders/therapy , Child , Combined Modality Therapy , Cooperative Behavior , Disease Progression , Early Diagnosis , Genes, Retinoblastoma/genetics , Genetic Testing , Humans , Interdisciplinary Communication , Neoplasm Staging , Neoplasms, Multiple Primary/diagnosis , Neoplasms, Multiple Primary/genetics , Neoplasms, Multiple Primary/mortality , Neoplasms, Multiple Primary/pathology , Neoplasms, Multiple Primary/therapy , Prognosis , Retinal Neoplasms/genetics , Retinal Neoplasms/mortality , Retinal Neoplasms/pathology , Retinoblastoma/genetics , Retinoblastoma/mortality , Retinoblastoma/pathology , Survival Rate , Vision Disorders/mortality , Vision Disorders/pathologyABSTRACT
BACKGROUND: Using mRNA expression-derived signatures as predictors of individual patient outcome has been a goal ever since the introduction of microarrays. Here, we addressed whether analyses of tumour mRNA at the exon level can improve on the predictive power and classification accuracy of gene-based expression profiles using neuroblastoma as a model. METHODS: In a patient cohort comprising 113 primary neuroblastoma specimens expression profiling using exon-level analyses was performed to define predictive signatures using various machine-learning techniques. Alternative transcript use was calculated from relative exon expression. Validation of alternative transcripts was achieved using qPCR- and cell-based approaches. RESULTS: Both predictors derived from the gene or the exon levels resulted in prediction accuracies >80% for both event-free and overall survival and proved as independent prognostic markers in multivariate analyses. Alternative transcript use was most prominently linked to the amplification status of the MYCN oncogene, expression of the TrkA/NTRK1 neurotrophin receptor and survival. CONCLUSION: As exon level-based prediction yields comparable, but not significantly better, prediction accuracy than gene expression-based predictors, gene-based assays seem to be sufficiently precise for predicting outcome of neuroblastoma patients. However, exon-level analyses provide added knowledge by identifying alternative transcript use, which should deepen the understanding of neuroblastoma biology.
Subject(s)
Exons/genetics , Neuroblastoma/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Receptor, trkA/genetics , Cell Line, Tumor , Child, Preschool , Gene Expression Profiling , Humans , Infant , N-Myc Proto-Oncogene Protein , Neuroblastoma/mortality , Prognosis , RNA, Messenger , Risk Factors , Survival AnalysisABSTRACT
Lysine (K)-specific demethylase 1A (LSD1/KDM1A) has been identified as a potential therapeutic target in solid cancers and more recently in acute myeloid leukemia. However, the potential side effects of a LSD1-inhibitory therapy remain elusive. Here, we show, with a newly established conditional in vivo knockdown model, that LSD1 represents a central regulator of hematopoietic stem and progenitor cells. LSD1 knockdown (LSD1-kd) expanded progenitor numbers by enhancing their proliferative behavior. LSD1-kd led to an extensive expansion of granulomonocytic, erythroid and megakaryocytic progenitors. In contrast, terminal granulopoiesis, erythropoiesis and platelet production were severely inhibited. The only exception was monopoiesis, which was promoted by LSD1 deficiency. Importantly, we showed that peripheral blood granulocytopenia, monocytosis, anemia and thrombocytopenia were reversible after LSD1-kd termination. Extramedullary splenic hematopoiesis contributed to the phenotypic reversion, and progenitor populations remained expanded. LSD1-kd was associated with the upregulation of key hematopoietic genes, including Gfi1b, Hoxa9 and Meis1, which are known regulators of the HSC/progenitor compartment. We also demonstrated that LSD1-kd abrogated Gfi1b-negative autoregulation by crossing LSD1-kd with Gfi1b:GFP mice. Taken together, our findings distinguish LSD1 as a critical regulator of hematopoiesis and point to severe, but reversible, side effects of a LSD1-targeted therapy.
Subject(s)
Cell Differentiation , Cell Proliferation , Hematopoiesis/physiology , Oxidoreductases, N-Demethylating/physiology , Stem Cells/cytology , Animals , Blotting, Western , Erythropoiesis/physiology , Female , Flow Cytometry , Granulocytes/cytology , Granulocytes/metabolism , Histone Demethylases , Humans , Integrases/metabolism , Male , Megakaryocytes/cytology , Megakaryocytes/metabolism , Mice , Mice, Transgenic , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Stem Cells/metabolismABSTRACT
Neuroblastoma tumors frequently show loss of heterozygosity of chromosome 11q with a shortest region of overlap in the 11q23 region. These deletions are thought to cause inactivation of tumor suppressor genes leading to haploinsufficiency. Alternatively, micro-deletions could lead to gene fusion products that are tumor driving. To identify such events we analyzed a series of neuroblastomas by comparative genomic hybridization and single-nucleotide polymorphism arrays and integrated these data with Affymetrix mRNA profiling data with the bioinformatic tool R2 (http://r2.amc.nl). We identified three neuroblastoma samples with small interstitial deletions at 11q23, upstream of the forkhead-box R1 transcription factor (FOXR1). Genes at the proximal side of the deletion were fused to FOXR1, resulting in fusion transcripts of MLL-FOXR1 and PAFAH1B2-FOXR1. FOXR1 expression has only been detected in early embryogenesis. Affymetrix microarray analysis showed high FOXR1 mRNA expression exclusively in the neuroblastomas with micro-deletions and rare cases of other tumor types, including osteosarcoma cell line HOS. RNAi silencing of FOXR1 strongly inhibited proliferation of HOS cells and triggered apoptosis. Expression profiling of these cells and reporter assays suggested that FOXR1 is a negative regulator of fork-head box factor-mediated transcription. The neural crest stem cell line JoMa1 proliferates in culture conditional to activity of a MYC-ER transgene. Over-expression of the wild-type FOXR1 could functionally replace MYC and drive proliferation of JoMa1. We conclude that FOXR1 is recurrently activated in neuroblastoma by intrachromosomal deletion/fusion events, resulting in overexpression of fusion transcripts. Forkhead-box transcription factors have not been previously implicated in neuroblastoma pathogenesis. Furthermore, this is the first identification of intrachromosomal fusion genes in neuroblastoma.
Subject(s)
Chromosomes, Human, Pair 11 , Neuroblastoma/genetics , Recombination, Genetic , Animals , Cell Line, Tumor , Comparative Genomic Hybridization , Gene Expression Regulation, Neoplastic , Gene Silencing , Haploinsufficiency , Humans , Loss of Heterozygosity , Mice , Oncogene Fusion , Polymorphism, Single Nucleotide , Sequence DeletionABSTRACT
PURPOSE: Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary. METHODS: We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC). RESULTS: Cohen's kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression. CONCLUSION: TH RT-qPCR alone is limited for detection of NB cells because of "false positives" in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.