Cohort Of Neuroblastoma Patients Research Articles

Untargeted LC-HRMS Based-Plasma Metabolomics Reveals 3-O-Methyldopa as a New Biomarker of Poor Prognosis in High-Risk Neuroblastoma.

Neuroblastoma (NB) is the most common extracranial malignant tumor in children. Although the survival rate of NB has improved over the years, the outcome of NB still remains poor for over 30% of cases. A more accurate risk stratification remains a key point in the study of NB and the availability of novel prognostic biomarkers of “high-risk” at diagnosis could help improving patient stratification and predicting outcome.In this paper we show a biomarker discovery approach applied to the plasma of 172 NB patients. Plasma samples from a first cohort of NB patients and age-matched healthy controls were used for untargeted metabolomics analysis based on high-resolution mass spectrometry (HRMS). Differential expression analysis highlighted a number of metabolites annotated with a high degree of identification. Among them, 3-O-methyldopa (3-O-MD) was validated in a second cohort of NB patients using a targeted metabolite profiling approach and its prognostic potential was also analyzed by survival analysis on patients with 3 years follow-up. High expression of 3-O-MD was associated with worse prognosis in the subset of patients with stage M tumor (log-rank p < 0.05) and, among them, it was confirmed as a prognostic factor able to stratify high-risk patients older than 18 months. 3-O-MD might be thus considered as a novel prognostic biomarker of NB eligible to be included at diagnosis among catecholamine metabolite panels in prospective clinical studies. Further studies are warranted to exploit other potential biomarkers highlighted using our approach.

Abstract 5403: Targeting death-receptors in therapy resistant progressive neuroblastoma: A preliminary insight

Abstract Neuroblastoma is the most common cancer in infants and accounts for 9.1% of childhood cancer-related deaths. Despite the current standard of care (SOC), drug-resistant neuroblastoma significantly compromises patient survival. Hence it is critical to identify new and less toxic therapeutic approaches for better patient outcomes. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which mediates its effect through the activation of the death receptor (DR) 4 and 5 are found on many cell types and are the main pathway for apoptosis. These can death receptors can kill most tumor cell types expressing TRAIL-receptor 1. Mining RNA-Seq data across multiple cohorts of neuroblastoma patients indicated that high levels of DR4 and DR5 significantly associated with poor clinical outcomes (OS, EFS, RFS). Further, examining the surface expression of DR4 and DR5 by immunohistochemistry using anti-human rabbit monoclonal antibodies on custom-archived TMAs for 100 NB patients revealed profound correlation with poor OS and RFS. Immunoblot analysis for DR4 and DR5 level in a panel of stage 4 patient-derived cell-lines during diagnosis and from progressive disease after therapy displayed high-levels of death receptor expression in progressive disease. Preliminary co-culture investigations with genetically engineered mesenchymal stem cells (MSCs) expressing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) mimicking targeted delivery showed heightened differentiation and death (limited dilution tumorosphere assay) of therapy resistant cells. Further these studies exhibited inhibition of tumor cell migration and invasion. The results demonstrate the benefit of targeted TRAIL delivery in in the treatment of therapy resistant neuroblastoma and further imply that the genetically engineered MSCs could serve as a clinically valuable tool for improved therapeutic strategy in this setting. Citation Format: Dinesh Babu Somasundaram, Andrew Maher, Sheeja Aravindan, Terence S. Herman, Natarajan Aravindan. Targeting death-receptors in therapy resistant progressive neuroblastoma: A preliminary insight [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5403.

The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma

Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13–15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

Abstract 4374: The role of glycine decarboxylase in neuroblastoma

Abstract Neuroblastoma is the most common cancer in infants. While the low and intermediate risk groups of neuroblastoma patients have ≥90% five-year-survival rates, the high-risk group has only 40-50% five-year-survival rates despite multimodal therapies. Genomic amplification of the oncogene MYCN (a member of the MYC family of transcription factors) is a major cause of high-risk neuroblastoma and is strongly correlated with poor prognosis. A key function of MYCN is to promote cell growth and proliferation, which requires increased cellular metabolism to meet the biosynthetic demand for growth. Serine-Glycine-One-Carbon (SGOC) metabolism is particularly important in sustaining cell proliferation by producing amino acids for protein synthesis and one-carbon units for nucleotide production. We investigated the role of glycine decarboxylase (GLDC) in neuroblastoma. GLDC catalyzes the first reaction in glycine cleavage, which generates one-carbon unit and reduced NADH. Analysis of gene expression data from two cohorts of neuroblastoma patients showed that GLDC expression is correlated with advanced stages, high-risk disease and poor prognosis in neuroblastoma. We found that GLDC is required for neuroblastoma cell proliferation as silencing GLDC expression by shRNA induced G1 cell cycle arrest. Consistent with this observation, microarray gene expression profiling revealed that GLDC knockdown resulted in a significant decrease in the expression of E2F target genes. In addition, we obtained evidence that GLDC is a direct target gene of MYCN. Knockdown of MYCN expression reduced GLDC mRNA and protein expression, and overexpression of MYCN increased GLDC mRNA and protein levels. Moreover, chromatin immunoprecipitation with quantitative PCR demonstrated that MYCN binds to the GLDC promoter region. These findings suggest an important role of GLDC in driving neuroblastoma cell proliferation, which could be exploited as a therapeutic strategy against high-risk neuroblastoma with MYCN amplification. Citation Format: Ahmet Alptekin, Jane Ding, Bingwei Ye, Han-Fei Ding. The role of glycine decarboxylase in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4374.

Легочные метастазы при нейробластоме у детей

Lung metastases in patients with neuroblastoma (NB) are an extremely rare situation. Such patients have a worse prognosis and more aggressive clinical behavior of the underlying disease. This article presents an analysis of the epidemiological, clinical, radiologic features and prognosis of the disease in a group of patients with stage 4 NB and lung metastases. The cohort of NB patients with stage 4 according to the International Neuroblastoma Staging System (INSS) treated in NRC PHOI named after Dmitry Rogachev for the period 01.2012–12.2016 (60 months) was included in the analysis. Patients were stratified and treated according to the modified protocol NB-2004. Comparative analysis of patients with high-risk NB with and without lung metastases was done. Estimated event-free and overall survival were determined using Kaplan–Meier methods. Among 132 patients with stage 4 NB lung metastases were detected in 10 cases, which represented 7.5% of the whole group. The median age of the patients with lung metastases was 26.4 months (range 4.3–92.5). 9 (90%) patients were stratified to highrisk group, 1 (10%) to intermediate risk group. Comparative analysis of patients with stage 4 high-risk NB with and without lung metastases showed no statistical difference in comparison by sex, age, localization of the primary tumor, MYCN status, and the number of metastatic compartments. Progression during induction therapy was more frequent in patients with lung metastases (p = 0.02). 2-year overall survival (OS) in the group of patients with lung metastases was lower than in group without metastases to the lung (50.0 ± 15.8% vs 75.9 ± 3.9%, p = 0.045). In our study, the presence of lung metastases was more common than was described elsewhere and is clinically important because it portends a poor prognosis.

IL-2 in the tumor microenvironment is necessary for Wiskott-Aldrich syndrome protein deficient NK cells to respond to tumors in vivo.

To kill target cells, natural killer (NK) cells organize signaling from activating and inhibitory receptors to form a lytic synapse. Wiskott-Aldrich syndrome (WAS) patients have loss-of-function mutations in the actin regulator WASp and suffer from immunodeficiency with increased risk to develop lymphoreticular malignancies. NK cells from WAS patients fail to form lytic synapses, however, the functional outcome in vivo remains unknown. Here, we show that WASp KO NK cells had decreased capacity to degranulate and produce IFNγ upon NKp46 stimulation and this was associated with reduced capacity to kill MHC class I-deficient hematopoietic grafts. Pre-treatment of WASp KO NK cells with IL-2 ex vivo restored degranulation, IFNγ production, and killing of MHC class I negative hematopoietic grafts. Moreover, WASp KO mice controlled growth of A20 lymphoma cells that naturally produced IL-2. WASp KO NK cells showed increased expression of DNAM-1, LAG-3, and KLRG1, all receptors associated with cellular exhaustion and NK cell memory. NK cells isolated from WAS patient spleen cells showed increased expression of DNAM-1 and had low to negative expression of CD56, a phenotype associated with NK cells exhaustion. Finally, in a cohort of neuroblastoma patients we identified a strong correlation between WASp, IL-2, and patient survival.

Abstract A01: Evaluation of hypoxia adaptation in neuroblastoma identifies reproducible transcriptional and phenotypic responses

Abstract Although metabolic adaptation is a cornerstone of the hypoxia response, very little is known about hypoxia-induced metabolic changes in neuroblastoma or how the metabolome influences phenotype. Identifying genes that regulate neuroblastoma metabolism and also influence clinical behavior may provide clues for novel therapeutic targets. Mixed linear models were used to identify differentially expressed genes between neuroblastoma cells grown in hypoxia or normoxia in two independent experiments (GEO accessions GSE17714 and GSE55391). Linear models were also used in to identify differentially expressed genes from tumors of two independent cohorts of neuroblastoma patients (GEO accession GSE16254 and EMBL accession E-MTAB-179) who survived compared to those that did not. We utilized a false discovery rate of 0.01 and fold change in the top or bottom 10% of all genes as a significance cutoff. qPCR was used to validate expression differences in multiple neuroblastoma cell lines. 157 genes, significantly exceeding chance (p &amp;lt; 1x10-6) on permutation testing, were present in both neuroblastoma cell line data sets, all but five of which showed consistent directionality of expression change from normoxia to hypoxia, including 44 involved in metabolism. These genes were enriched for hypoxia and metabolic pathways: HIF-1α transcription factor network (p = 1.3x10-11), glycolysis (p = 1.9x10-8) and fructose metabolism (p = 1.0x10-3). 826 genes, significantly exceeding chance (p &amp;lt; 1x10-6) were differentially expressed in both patient cohorts of 478 and 88 patients. These genes were enriched for cell cycle pathways (p = 4.5x10-7). Of these genes, nine of them were also differentially expressed in hypoxia compared to normoxia with consistent directionality, again more than expected by chance (p &amp;lt; 1x10-6). High expression in eight of the nine genes was also significantly associated with poor outcome in a Kaplan-Meier analysis of both of the patient cohorts evaluated. Three of these genes are part of the glycolytic pathway and three more are directly involved in cellular metabolism. In the SK-N-BE2 cell line, all eight of our identified genes are up-regulated in hypoxia (p &amp;lt; 0.05). Analysis of the LAN-5, La1-55n, SK-N-DZ cell lines and show similar results. MTT assays of proliferation show the expected decreased proliferation for all of these cell lines in 48 hours of hypoxia compared to normoxia (p &amp;lt; 0.05). Analysis of cell cycle by flow cytometry in SK-N-BE2 cells shows an increase of cells in G0/G1 in normoxia compared to hypoxia (58.3% vs. 71.5% p = 0.04) and decrease of cells in S phase (24.5% vs. 13.6%, p = 0.001). We have identified eight genes with increased expression in hypoxia and are associated with poor survival in patients. Efforts to use shRNA to alter cellular phenotype are ongoing. Citation Format: Mark A. Aplebaum, Aashish Jha, Alexandre Chlenski, Christopher Mariani, Clara Kao, Mildred Nelson, Kyle Hernandez, Helen Salwen, Marija Dobratic, Kevin White, Barbara Stranger, Susan L. Cohn. Evaluation of hypoxia adaptation in neuroblastoma identifies reproducible transcriptional and phenotypic responses. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A01.

Abstract 1626: Evaluation of the expression of neuroblastoma-associated genes for bone marrow (BM) involvement and minimal residual disease (MRD) detection

Abstract Methods. Panel of 4 tumor-associated genes (TAGs): PHOX2B, TH, ELAVL4 and GD2 was created. Normalized expression (CtABL-CtTAG) of these genes was analyzed in neuroblastoma (NB) cell lines, in 26 BM samples from patients without malignancies, in the discovery (331 BM samples from 57 patients) and validation cohorts of NB patients (311 BM samples from 55 patients) and in 23 peripheral blood stem cells (PBSC) preparations. For performing of ROC analysis BM samples were defined as positive in case of detectable PHOX2B expression or tumor cells presence in BM smears. Threshold levels (TL) of each gene expression were established and subsequently applied for overall correct prediction (OCP) computation. Prognostic significance of the BM positivity for the evaluated markers was determined using event-free (EFS) and overall survival (OS) rates with median of follow-up time 2.45 years. Results. Neither PHOX2B nor TH expression was detected in the intact BM, while expression of ELAVL4 was revealed in 20 and GD2 - in 15 of 26 BM samples of patients without malignancies. In the discovery cohort 105 analyzed BM samples were positive for PHOX2B expression, while 101 for TH. TH expression was revealed in 5 out of 224 negative BM samples. ELAVL4 and GD2 expression was detected in all 107 positive BM samples as well as in the majority of negative BM samples (209 and 197 of 224, respectively). TL of TH, ELAVL4 and GD2 expression established by ROC analysis were -16.535, -7.130 and -8.459. OCP values calculated using TL achieved 0.952, 0.828 and 0.767 correspondingly. OCP for PHOX2B was 0.994. Due to high OCP values for PHOX2B and TH expression of these genes was analyzed in the validation cohort, where TL for TH was -13.995, while OCP values achieved 0.997 for PHOX2B and 0.939 for TH. Presence of PHOX2B/TH expression in BM at the time of primary NB diagnostics led to decreased both EFS (.31SE.12 vs .81SE.06, p&amp;lt;.001) and OS (.31SE.13 vs .87SE.05, p&amp;lt;.001). Persistence of TAGs expression during the treatment demonstrated trend to reduced EFS (.27SE.12 vs .43SE.19, p = .08). No expression of TAGs was find in the PBSC preparations while positivity for PHOX2B/TH expression before PBSC apheresis had strongly negative prognostic impact (EFS .00 vs .35SE.14, p = .04; OS .00 vs .36SE.15, p = .03) despite of CD34+ selection. Predominance of PHOX2B expression over TH in BM of primary NB patients with the difference in normalized expression greater then 1.68 had negative prognostic significance: EFS .00 vs .56SE.12, p = .017, OS .00 vs .72SE.11, p = .006. Conclusion. PHOX2B and TH are the most appropriate markers for BM involvement and MRD detection in NB patients. Presence of these TAGs expression in the BM at the time of primary diagnostics and before PBSC apheresis had strongly negative prognostic impact. Predominance of PHOX2B expression over TH in the BM can define high risk patients. Citation Format: Alexander E. Druy, Egor V. Shorikov, Grigory A. Tsaur, Alexander M. Popov, Leonid I. Saveliev, Larisa G. Fechina. Evaluation of the expression of neuroblastoma-associated genes for bone marrow (BM) involvement and minimal residual disease (MRD) detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1626. doi:10.1158/1538-7445.AM2015-1626

Abstract 5138: Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1

Abstract Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. We aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis.Gene and protein expression was analysed by real-time RT-PCR and immunoblot. Cell survival/death was examined by flow cytometry study of Annexin V-staining. The prognostic value of TP53INP1 expression in tumor tissues was investigated in three independent cohorts of neuroblastoma patients. TP53INP1 was one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma. Citation Format: Jeyran Shahbazi, Christopher J. Scarlett, Murray Norris, Bing Liu, Michelle Haber, Andrew E. Tee, Alice Carrier, Andrew V. Biankin, Wendy B. London, Glenn M. Marshall, Richard Lock, Tao Liu. Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5138. doi:10.1158/1538-7445.AM2014-5138

Effects of a novel long noncoding RNA, lncUSMycN, on N-Myc expression and neuroblastoma progression.

Patients with neuroblastoma due to the amplification of a 130-kb genomic DNA region containing the MYCN oncogene have poor prognoses. Bioinformatics data were used to discover a novel long noncoding RNA, lncUSMycN, at the 130-kb amplicon. RNA-protein pull-down assays were used to identify proteins bound to lncUSMycN RNA. Kaplan-Meier survival analysis, multivariable Cox regression, and two-sided log-rank test were used to examine the prognostic value of lncUSMycN and NonO expression in three cohorts of neuroblastoma patients (n = 47, 88, and 476, respectively). Neuroblastoma-bearing mice were treated with antisense oligonucleotides targeting lncUSMycN (n = 12) or mismatch sequence (n = 13), and results were analyzed by multiple comparison two-way analysis of variance. All statistical tests were two-sided. Bioinformatics data predicted lncUSMycN gene and RNA, and reverse-transcription polymerase chain reaction confirmed its three exons and two introns. The lncUSMycN gene was coamplified with MYCN in 88 of 341 human neuroblastoma tissues. lncUSMycN RNA bound to the RNA-binding protein NonO, leading to N-Myc RNA upregulation and neuroblastoma cell proliferation. High levels of lncUSMycN and NonO expression in human neuroblastoma tissues independently predicted poor patient prognoses (lncUSMycN: hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.06 to 3.28, P = .03; NonO: HR = 2.48, 95% CI = 1.34 to 4.57, P = .004). Treatment with antisense oligonucleotides targeting lncUSMycN in neuroblastoma-bearing mice statistically significantly hindered tumor progression (P < .001). Our data demonstrate the important roles of lncUSMycN and NonO in regulating N-Myc expression and neuroblastoma oncogenesis and provide the first evidence that amplification of long noncoding RNA genes can contribute to tumorigenesis.

Plasma Levels of Soluble HLA-E and HLA-F at Diagnosis May Predict Overall Survival of Neuroblastoma Patients

The purpose of this study was to identify the plasma/serum biomarkers that are able to predict overall survival (OS) of neuroblastoma (NB) patients. Concentration of soluble (s) biomarkers was evaluated in plasma (sHLA-E, sHLA-F, chromogranin, and B7H3) or serum (calprotectin) samples from NB patients or healthy children. The levels of biomarkers that were significantly higher in NB patients were then analyzed considering localized or metastatic subsets. Finally, biomarkers that were significantly different in these two subsets were correlated with patient's outcome. With the exception of B7H3, levels of all molecules were significantly higher in NB patients than those in controls. However, only chromogranin, sHLA-E, and sHLA-F levels were different between patients with metastatic and localized tumors. sHLA-E and -F levels correlated with each other but not chromogranin. Chromogranin levels correlated with different event-free survival (EFS), whereas sHLA-E and -F levels also correlated with different OS. Association with OS was also detected considering only patients with metastatic disease. In conclusion, low levels of sHLA-E and -F significantly associated with worse EFS/OS in the whole cohort of NB patients and in patients with metastatic NB. Thus, these molecules deserve to be tested in prospective studies to evaluate their predictive power for high-risk NB patients.

Aurora A is a negative prognostic factor and a new therapeutic target in human neuroblastoma

We studied expression of the Aurora A gene and its clinical significance in a cohort of neuroblastoma patients. In addition, we investigated the antitumor activity of MLN8054, a novel small-molecule inhibitor of Aurora A kinase, on cultured NB cell lines in vitro. Aurora A mRNA expression was assessed by quantitative real-time PCR in tumor tissue specimens from 67 patients at diagnosis and in 9 human neuroblastoma cell lines. Western blot assays for Aurora A protein were done on tumor tissue of 53 patients. The results were correlated with various prognostic factors of neuroblastoma. Aurora A mRNA and protein expression were identified in 9 of 9 neuroblastoma cell lines. Overexpression of Aurora A mRNA in neuroblastoma tumor tissue is associated with high risk (P = 0.019), high-stage (International Neuroblastoma Staging System III and IV) tumors (P = 0.007), unfavorable histology (P = 0.007), MYCN amplification (P = 0.017), disease relapse (P = 0.019), and decreased progression-free survival (P < 0.0001) but not correlated with the age at diagnosis (P = 0.877). Similarly, Aurora A protein expression also significantly correlated with high risk (P = 0.011), high stage (P = 0.0028), unfavorable histology (P = 0.0006), MYCN amplification (P = 0.0029), and disease relapse (P = 0.044). Small interfering RNA-mediated knockdown of the endogenous Aurora A gene causes a proliferation defect and enhances chemosensitivity in human neuroblastoma cell lines. In support of these observations, the Aurora A kinase inhibitor, MLN8054, markedly inhibited growth of cultured neuroblastoma cell lines through an apoptosis-dependent pathway. Overexpression of Aurora A is associated with disease progression in neuroblastoma. Inhibition of this kinase is a promising modality for neuroblastoma treatment.

Genes involved in regulation of stem cell properties: studies on their expression in a small cohort of neuroblastoma patients

Cancer stem cells have been isolated from many tumors. Several evidences prove that neuroblastoma contains its own stem cell-like cancer cells. We chose to analyze 20 neuroblastoma tumor samples in the expression of 13 genes involved in the regulation of stem cell properties to evaluate if their misregulation could have a clinical relevance. In several specimens we detected the expression of genes belonging to the OCT3/SOX2/NANOG/KLF4 core circuitry that acts at the highest level in regulating stem cell biology. This result is in agreement with studies showing the existence of malignant stem cells in neuroblastoma. We also observed differences in the expression of some stemness-related genes that may be useful for developing new prognostic analyses. In fact, preliminary data suggests that the presence/absence of UTF1 along with differences in BMI1 mRNA levels could distinguish low grade neuroblastomas from IV stage tumors.

Angiogenesis in neuroblastoma: relationship to survival and other prognostic factors in a cohort of neuroblastoma patients.

To study angiogenesis in neuroblastoma, using morphometric and computerized image analysis, and correlate the results with survival and other prognostic factors. Sixty-nine patients from the Spanish Cooperative Study for Neuroblastoma were studied. Tumoral angiogenesis was studied using an avidin-biotin immunoperoxidase technique with an anti-CD34 antibody. Vascular parameters (VPs) were analyzed by a computerized system. Statistical analysis was also performed. Sixty-six samples had adequate tumoral tissue, and their tumoral vessels were counted. Endothelial cells were more prominent in pure neuroblastomas than in maturing and more mature tumors. VPs showed no statistical difference between the groups of patients as defined by the levels of the other prognostic factors in neuroblastoma: age, stage, histopathology, TRK-A, P-glycoprotein expression, or MYCN copy number. In patients who relapsed, tumors did not show statistically significant difference in VPs when compared with tumors from patients who did not relapse. There was also no difference in VPs in tumors from living patients when compared with tumors from deceased patients. Overall survival was 75%, and event-free survival was 55% at 50 months. VPs could be adequately determined by a computerized system in neuroblastoma; however, VPs were not predictive of survival for our patients. In our patients, neither disseminated nor local relapses were influenced by the angiogenic characteristics of the tumors.