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Neuroblastoma (NB) is a pediatric tumor of embryonic origin. About 1–2% of all NBs are familial cases, and genetic predisposition is suspected for the remaining cases. During the last decade, genome-wide association studies (GWAS) and high-throughput sequencing approaches have been used to identify associations among common and rare genetic variants and NB risk. Substantial data has been produced by large patient cohorts that implicate various genes in NB tumorigenesis, such as CASC15, BARD1, CHEK2, LMO1, LIN28B, AXIN2, BRCA1, TP53, SMARCA4, and CDK1NB. NB, as well as other pediatric cancers, has few recurrent mutations but several copy number variations (CNVs). Almost all NBs show both numerical and structural CNVs. The proportion between numerical and structural CNVs differs between localized and metastatic tumors, with a greater prevalence of structural CNVs in metastatic NB. This genomic chaos frequently identified in NBs suggests that chromosome instability (CIN) could be one of the major actors in NB oncogenesis. Interestingly, many NB-predisposing variants occur in genes involved in the control of genome stability, mitosis, and normal chromosome separation. Here, we discuss the relationship between genetic predisposition and CIN in NB. [ABSTRACT FROM AUTHOR]

Neuroblastoma is a neural crest-derived tumor that accounts for 7–10% of all malignancies in children and ~15% of all childhood cancer-associated mortalities. Approximately 50% of patients are characterized as high-risk (HR) and have an overall survival of <40% at 5 years from diagnosis. HR patients with unfavorable prognosis exhibit several structural copy number variations (CNVs), whereas localized tumors belonging to patients in the low- and intermediate-risk classes, have favorable outcomes and display several numerical CNVs. Taken together these results are indicative of chromosome instability (CIN) in neuroblastoma tumor cells. The present review discusses multiple aspects of CIN including methods of measuring CIN, CIN targeting as a therapeutic strategy in cancer and the effects of CIN in neuroblastoma development and aggressiveness with particular emphasis on the CIN gene signature associated with HR neuroblastoma patients. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is an embryonic malignancy of the sympathetic nervous system with heterogeneous biological, morphological, genetic and clinical characteristics. Although genomic studies revealed the specific biological features of NB pathogenesis useful for new therapeutic approaches, the improvement of high‐risk (HR)‐NB patients overall survival remains unsatisfactory. To further clarify the biological basis of disease aggressiveness, we used whole‐exome sequencing to examine the genomic landscape of HR‐NB patients at stage M with short survival (SS) and long survival (LS). Only a few genes, including SMARCA4, SMO, ZNF44 and CHD2, were recurrently and specifically mutated in the SS group, confirming the low recurrence of common mutations in this tumor. A systems biology approach revealed that in the two patient groups, mutations occurred in different pathways. Mutated genes (ARHGEF11, CACNA1G, FGF4, PTPRA, PTK2, ANK3, SMO, NTNG2, VCL and NID2) regulate the MAPK pathway associated with the organization of the extracellular matrix, cell motility through PTK2 signaling and matrix metalloproteinase activity. Moreover, we detected mutations in LAMA2, PTK2, LAMA4, and MMP14 genes, impairing MET signaling, in SFI1 and CHD2 involved in centrosome maturation and chromosome remodeling, in AK7 and SPTLC2, which regulate the metabolism of nucleotides and lipoproteins, and in NALCN, SLC12A1, SLC9A9, which are involved in the transport of small molecules. Notably, connected networks of somatically mutated genes specific for SS patients were identified. The detection of mutated genes present at the onset of disease may help to address an early treatment of HR‐NB patients using FDA‐approved compounds targeting the deregulated pathways. What's new? Most patients with metastatic neuroblastoma don't survive 5 years from diagnosis, despite responding well to first‐line treatments. Previous work comparing short‐survival and long‐survival patients identified some key chromosomal differences. These authors take the search deeper, conducting whole‐exome sequencing to compare somatic mutations between patients who survived at least 5 years and those who did not. They determined that mutations among the short‐survival group affected different pathways than those afflicting the long‐survival patients. In some cases, drugs already exist that target these proteins, suggesting that testing for these mutations at the time of diagnosis could indicate specific treatments. [ABSTRACT FROM AUTHOR]

Neuroblastoma is an embryonic malignancy of early childhood originating from neural crest cells and showing heterogeneous biological, morphological, genetic and clinical characteristics. The correct stratification of neuroblastoma patients within risk groups (low, intermediate, high and ultra-high) is critical for the adequate treatment of the patients. High-throughput technologies in the Omics disciplines are leading to significant insights into the molecular pathogenesis of neuroblastoma. Nonetheless, further study of Omics data is necessary to better characterise neuroblastoma tumour biology. In the present review, we report an update of compounds that are used in preclinical tests and/or in Phase I-II trials for neuroblastoma. Furthermore, we recapitulate a number of compounds targeting proteins associated to neuroblastoma: MYCN (direct and indirect inhibitors) and downstream targets, Trk, ALK and its downstream signalling pathways. In particular, for the latter, given the frequency of ALK gene deregulation in neuroblastoma patients, we discuss on second-generation ALK inhibitors in preclinical or clinical phases developed for the treatment of neuroblastoma patients resistant to crizotinib. We summarise how Omics drive clinical trials for neuroblastoma treatment and how much the research of biological targets is useful for personalised medicine. Finally, we give an overview of the most recent druggable targets selected by Omics investigation and discuss how the Omics results can provide us additional advantages for overcoming tumour drug resistance. [ABSTRACT FROM AUTHOR]

Background: Neuroblastoma is a pediatric cancer with a low survival rate of patients with metastatic stage 4 disease. Tumor aggressiveness and progression have been associated with structural copy number variations (CNVs) that are observed in malignant cells. In contrast, localized Neuroblastomas, which are associated with a low number of structural CNVs but frequent numerical CNVs, are less aggressive, and patients have good outcomes. Finally, whole-genome and whole-exome sequencing of Neuroblastoma tissues have shown few damaging mutations in these tumors.Conclusions: In the present report it is proposed that chromosome instability (CIN) plays a major role in Neuroblastoma tumorigenesis and that CIN is already present in the early phases of tumor development. High CIN can promote several types of chromosomal damage including chromothripsis, gene deletion, amplification and rearrangements, which deregulate gene expression. Indeed, gene rearrangements have been reported as a new scenario in the development of Neuroblastoma, which supports the hypothesis that CIN is an early step preliminary to the late catastrophic events leading to tumor development. [ABSTRACT FROM AUTHOR]

Neuroblastoma is a tumor arising in the peripheral sympathetic nervous system and is the most common cancer in childhood. Since most of the cellular and molecular mechanisms underlying neuroblastoma onset and progression remain unknown, the generation of new in vivo models might be appropriate to better dissect the peripheral sympathetic nervous system development in both physiological and disease states. This review is focused on the use of zebrafish as a suitable and innovative model to study neuroblastoma development. Here, we briefly summarize the current knowledge about zebrafish peripheral sympathetic nervous system formation, focusing on key genes and cellular pathways that play a crucial role in the differentiation of sympathetic neurons during embryonic development. In addition, we include examples of how genetic changes known to be associated with aggressive neuroblastoma can mimic this malignancy in zebrafish. Thus, we note the value of the zebrafish model in the field of neuroblastoma research, showing how it can improve our current knowledge about genes and biological pathways that contribute to malignant transformation and progression during embryonic life. [ABSTRACT FROM AUTHOR]

A growing field of evidence suggests the involvement of oncogenic receptor tyrosine kinases (RTKs) in the transformation of malignant cells. Constitutive and abnormal activation of RTKs may occur in tumors either through hyperactivation of mutated RTKs or via functional upregulation by RTK-coding gene amplification. In several types of cancer prognosis and therapeutic responses were found to be associated with deregulated activation of one or more RTKs. Therefore, targeting various RTKs remains a significant challenge in the treatment of patients with diverse malignancies. However, a frequent issue with the use of RTK inhibitors is drug resistance. Autophagy activation during treatment with RTK inhibitors has been commonly observed as an obstacle to more efficacious therapy and has been associated with the limited efficacy of RTK inhibitors. In the present review, we discuss autophagy activation after the administration of RTK inhibitors and summarize the achievements of combination RTK/autophagy inhibitor therapy in overcoming the reported resistance to RTK inhibitors in a growing number of cancers. [ABSTRACT FROM AUTHOR]

Background: The prognosis of children with metastatic stage 4 neuroblastoma (NB) has remained poor in the past decade.Patients and Methods: Using microarray analyses of 342 primary tumors, we here developed and validated an easy to use gene expression-based risk score including 18 genes, which can robustly predict the outcome of stage 4 patients.Results: This classifier was a significant predictor of overall survival in two independent validation cohorts [cohort 1 (n = 214): P = 6.3 × 10(-5); cohort 2 (n = 27): P = 3.1 × 10(-2)]. The prognostic value of the risk score was validated by multivariate analysis including the established markers age and MYCN status (P = 0.027). In the pooled validation cohorts (n = 241), integration of the risk score with the age and/or MYCN status identified subgroups with significantly differing overall survival (ranging from 35 to 100 %).Conclusion: Together, the 18-gene risk score classifier can identify patients with stage 4 NB with favorable outcome and may therefore improve risk assessment and treatment stratification of NB patients with disseminated disease. [ABSTRACT FROM AUTHOR]

Data from two contexts, i.e. the European Unresectable Neuroblastoma (EUNB) clinical trial and results from comparative genomic hybridisation (CGH) analyses from corresponding tumour samples shall be provided to existing repositories for secondary use. Utilizing the European Unified Patient IDentity Management (EUPID) as developed in the course of the ENCCA project, the following processes were applied to the data: standardization (providing interoperability), pseudonymization (generating distinct but linkable pseudonyms for both contexts), and linking both data sources. The applied procedures resulted in a joined dataset that did not contain any identifiers that would allow to backtrack the records to either data sources. This provided a high degree of privacy to the involved patients as required by data protection regulations, without preventing proper analysis. [ABSTRACT FROM AUTHOR]

Background: Metastatic neuroblastoma (NB) occurs in pediatric patients as stage 4S or stage 4 and it is characterized by heterogeneous clinical behavior associated with diverse genotypes. Tumors of stage 4 contain several structural copy number aberrations (CNAs) rarely found in stage 4S. To date, the NB tumorigenesis is not still elucidated, although it is evident that genomic instability plays a critical role in the genesis of the tumor. Here we propose a mathematical approach to decipher genomic data and we provide a new model of NB metastatic tumorigenesis. Method: We elucidate NB tumorigenesis using Enhanced Fused Lasso Latent Feature Model (E-FLLat) modeling the array comparative chromosome hybridization (aCGH) data of 190 metastatic NBs (63 stage 4S and 127 stage 4). This model for aCGH segmentation, based on the minimization of functional dictionary learning (DL), combines several penalties tailored to the specificities of aCGH data. In DL, the original signal is approximated by a linear weighted combination of atoms: the elements of the learned dictionary. Results: The hierarchical structures for stage 4S shows at the first level of the oncogenetic tree several whole chromosome gains except to the unbalanced gains of 17q, 2p and 2q. Conversely, the high CNA complexity found in stage 4 tumors, requires two different trees. Both stage 4 oncogenetic trees are marked diverged, up to five sublevels and the 17q gain is the most common event at the first level (2/3 nodes). Moreover the 11q deletion, one of the major unfavorable marker of disease progression, occurs before 3p loss indicating that critical chromosome aberrations appear at early stages of tumorigenesis. Finally, we also observed a significant (p = 0.025) association between patient age and chromosome loss in stage 4 cases. Conclusion: These results led us to propose a genome instability progressive model in which NB cells initiate with a DNA synthesis uncoupled from cell division, that leads to stage 4S tumors, primarily characterized by numerical aberrations, or stage 4 tumors with high levels of genome instability resulting in complex chromosome rearrangements associated with high tumor aggressiveness and rapid disease progression. [ABSTRACT FROM AUTHOR]

The prognosis of children with metastatic neuroblastoma (NB) > 18 months at diagnosis is dismal. Since the immune status of the tumor microenvironment could play a role in the history of disease, we evaluated the expression of CD45, CD14, ARG1, CD163, CD4, FOXP3, Perforin-1 (PRF1), Granzyme B (GRMB), and IL-10 mRNAs in primary tumors at diagnosis from children with metastatic NB and tested whether the transcript levels are significantly associated to event-free and overall survival (EFS and OS, resp.). Children with high expression of CD14, ARG1 and FOXP3 mRNA in their primary tumors had significantly better EFS. Elevated expression of CD14, and FOXP3 mRNA was significantly associated to better OS. CD14 mRNA expression levels significantly correlated to all markers, with the exception of CD4. Strong positive correlations were found between PRF1 and CD163, as well as between PFR1 and FOXP3. It is worth noting that the combination of high levels of CD14, FOXP3, and ARG1 mRNAs identified a small group of patients with excellent EFS and OS, whereas low levels of CD14 were sufficient to identify patients with dismal survival. Thus, the immune status of the primary tumors of high-risk NB patients may influence the natural history of this pediatric cancer. [ABSTRACT FROM AUTHOR]

Background: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis. Patients and Methods: We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays. Results and Conclusion: We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8 gene SNP D302H was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors. [ABSTRACT FROM AUTHOR]

Chemotherapy induces apoptosis and tumor regression primarily through activation of p53-mediated transcription. Neuroblastoma is a p53 wild type malignancy at diagnosis and repression of p53 signaling plays an important role in its pathogenesis. Recently developed small molecule inhibitors of the MDM2-p53 interaction are able to overcome this repression and potently activate p53 dependent apoptosis in malignancies with intact p53 downstream signaling. We used the small molecule MDM2 inhibitor, Nutlin-3a, to determine the p53 drug response signature in neuroblastoma cells. In addition to p53 mediated apoptotic signatures, GSEA and pathway analysis identified a set of p53-repressed genes that were reciprocally over-expressed in neuroblastoma patients with the worst overall outcome in multiple clinical cohorts. Multifactorial regression analysis identified a subset of four genes (CHAF1A, RRM2, MCM3, and MCM6) whose expression together strongly predicted overall and event-free survival (p<0.0001). The expression of these four genes was then validated by quantitative PCR in a large independent clinical cohort. Our findings further support the concept that oncogene-driven transcriptional networks opposing p53 activation are essential for the aggressive behavior and poor response to therapy of high-risk neuroblastoma. [ABSTRACT FROM AUTHOR]

Background: Common variants in DNA may predispose to onset and progression of neuroblastoma (NB). The genotype GG of single nucleotide polymorphism (SNP) rs1800795 (−174 G>C) in interleukin (IL)-6 promoter has been associated with lower survival of high-risk NB. Result: To evaluate the impact of IL-6 SNP rs1800795 on disease risk and phenotype, we analyzed 326 Italian NB patients and 511 controls. Moreover, we performed in silico and quantitative Real Time (qRT)-PCR analyses to evaluate the influence of the SNP on gene expression in 198 lymphoblastoid cell lines (LCLs) and in 31 NB tumors, respectively. Kaplan-Meier analysis was used to verify the association between IL-6 gene expression and patient survival. We found that IL-6 SNP is not involved in susceptibility to NB development. However, our results show that a low frequency of genotype CC is significantly associated with a low overall survival, advanced stage, and high-risk phenotype. The in silico (p = 2.61×10−5) and qRT-PCR (p = 0.03) analyses showed similar trend indicating that the CC genotype is correlated with increased level of IL-6 expression. In report gene assay, we showed that the −174 C variant had a significantly increased transcriptional activity compared with G allele (p = 0.0006). Moreover, Kaplan-Meier analysis demonstrated that high levels of IL-6 are associated with poor outcome in children with NB in two independent gene expression array datasets. Conclusions: The biological effect of SNP IL-6–174 G>C in relation to promotion of cancer progression is consistent with the observed decreased survival time. The present study suggests that SNP IL-6–174 G>C may be a useful marker for NB prognosis. [ABSTRACT FROM AUTHOR]

Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum consisting of four distinct subgroups: WNT, SHH, Group 3 and Group 4, which exhibit different molecular phenotypes. We studied the expression of Dickkopf (DKK) 1-4 family genes, inhibitors of the Wnt signaling cascade, in MB by screening 355 expression profiles derived from four independent datasets. Upregulation of DKK1, DKK2 and DKK4 mRNA was observed in the WNT subgroup, whereas DKK3 was downregulated in 80% MBs across subgroups with respect to the normal cerebellum (p < 0.001). Since copy number aberrations targeting the DKK3 locus (11p15.3) are rare events, we hypothesized that epigenetic factors could play a role in DKK3 regulation. Accordingly, we studied 77 miRNAs predicting to repress DKK3; however, no significant inverse correlation between miRNA/mRNA expression was observed. Moreover, the low methylation levels in the DKK3 promoters (median: 3%, 5% and 5% for promoter 1, 2 and 3, respectively) excluded the downregulation of gene expression by methylation. On the other hand, the treatment of MB cells with Trichostatin A (TSA), a potent inhibitor of histone deacetylases (HDAC), was able to restore both DKK3 mRNA and protein. In conclusion, DKK3 downregulation across all MB subgroups may be due to epigenetic mechanisms, in particular, through chromatin condensation. [ABSTRACT FROM AUTHOR]

Several neuroblastoma (NB) susceptibility loci have been identified within LINC00340, BARD1, LMO1, DUSP12, HSD17B12, DDX4, IL31RA, HACE1 and LIN28B by genome-wide association (GWA) studies including European American individuals. To validate and comprehensively evaluate the impact of the identified NB variants on disease risk and phenotype, we analyzed 16 single nucleotide polymorphisms (SNPs) in an Italian population (370 cases and 809 controls). We assessed their regulatory activity on gene expression in lymphoblastoid (LCLs) and NB cell lines. We evaluated the cumulative effect of the independent loci on NB risk and high-risk phenotype development in Italian and European American (1627 cases and 2575 controls) populations. All NB susceptibility genes replicated in the Italian dataset except for DDX4 and IL31RA, and the most significant SNP was rs6435862 in BARD1 (P = 8.4×10–15). BARD1 showed an additional and independent SNP association (rs7585356). This variant influenced BARD1 mRNA expression in LCLs and NB cell lines. No evidence of epistasis among the NB-associated variants was detected, whereas a cumulative effect of risk variants on NB risk (European Americans: Ptrend = 6.9×10–30, Italians: Ptrend = 8.55×1013) and development of high-risk phenotype (European Americans: Ptrend = 6.9×10–13, Italians: Ptrend = 2.2×10–1) was observed in a dose-dependent manner. These results provide further evidence that the risk loci identified in GWA studies contribute to NB susceptibility in distinct populations and strengthen the role of BARD1 as major genetic contributor to NB risk. This study shows that even in the absence of interaction the combination of several low-penetrance alleles has potential to distinguish subgroups of patients at different risks of developing NB. [ABSTRACT FROM AUTHOR]

About 50% of children with neuroblastoma (NB) show a metastatic disease and have a poor prognosis. However, disease progression is greatly variable and depends on patients' age and MYCN oncogene amplification. To investigate the role of patients' age in tumor aggressiveness, we performed array-CGH and gene expression profiles of three groups (G) of metastatic NBs: G1, stage 4S patients and MYCN single copy ( MYCN−) tumors; G2, stage 4 patients, ≤18 months of age, MYCN− tumors and favorable outcome and G3, Stage 4 patients, ≥19 months with unfavorable outcome. G1 was characterized by numerical aberrations prevalently; on the contrary, all G3 tumors had structural rearrangements, whereas G2 showed an intermediate pattern. The average of numerical alterations decreased significantly from G1 to G2 to G3 ( p < 0.01). Contrarily, the number of structural aberrations increased from G1 to G2 to G3 ( p < 2.35 E−05). Noteworthy, G3/ MYCN− NBs were characterized by several complex intrachromosome rearrangements. Expression analysis of the three groups showed significant differences in genes of Rho and Ras signaling pathways, development and adhesion, cell cycle regulation and telomerase activity. Accumulation of structural alterations increased with patients' age and was associated with a more aggressive disease. Abnormal expression of genes involved in cell cycle and telomerase in G3 may be responsible for the genomic instability in this cohort of patients. The higher DNA instability observed in G3/ MYCN− NBs than in MYCN-amplified G3 may also explain why patients ≥19 months have a poor outcome independently by MYCN status. [ABSTRACT FROM AUTHOR]

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients. [ABSTRACT FROM AUTHOR]

Abstract: Dicer is a ribonuclease playing a key role in the biogenesis of microRNAs and small interfering RNAs. Here we report the identification of a novel splice variant of human dicer gene, the first one bearing a modified coding sequence. It encodes a truncated protein, t-Dicer that lacks the dsRNA-binding domain and is defective in one of the two RNase III catalytic centers. The splice variant was found in neuroblastoma cells and in cells induced to neuronal differentiation, whereas it was not detectable in other cell lines or in normal tissues. Interestingly, it occurred in primary neuroblastic tumors, mainly in stroma poor neuroblastomas. [ABSTRACT FROM AUTHOR]

Background: Ultra Conserved Regions (UCRs) are a class of 481 noncoding sequences located in both intra- and inter-genic regions of the genome. The recent findings that they are significantly altered in adult chronic lymphocytic leukemias, carcinomas, and pediatric neuroblastomas lead to the hypothesis that UCRs may play a role in tumorigenesis. Results: We present a novel application of Ribo-SPIA™ isothermal linear amplification of minute RNA quantities for quantifying Transcribed-UCR (T-UCR) expression by quantitative PCR. Direct comparison of non-amplified with amplified cDNA in two neuroblastoma cell lines showed that the amplification approach increases sensitivity and repeatability in T-UCR quantification. It is noteworthy that the Ribo-SPIA™ step allowed us to analyze all 481 T-UCRs by using 150 ng of RNA, while introducing a minimal bias and preserving the magnitude of relative expression. Only the less abundant T-UCRs have high intra-assay variability, consistently with the Poisson distribution statistics and stochastic effects on PCR repeatability. Conclusions: We demonstrated that the quantification procedure shown here is an accurate and reliable technique for genome-wide non coding gene (i.e., UCRs) profiling using small amounts of RNA. This issue is particularly important because studies of transcription regulation are increasingly conducted in small homogeneous samples, such as laser capture microdissected or sorted cell populations. [ABSTRACT FROM AUTHOR]

The article presents a study to identify low intratumoral gene expression heterogeneity in neuroblastic tumors (NTs). It relates that gene expression data were analyzed by the Significance Analysis of Microarrays (SAM) and Game Theory (GT) methods. Accordingly, it shows that stroma-poor NTs (NTs-SP) and stroma-rich NTs (NTs-SR) gene signatures differ for a set of genes involved in distinct pathways, and low intratumoral heterogeneity at the messenger RNA (mRNA) level in both NT-SP and NT-SR.

Individual response to oxidative stress, due to exposure to asbestos fibres plays a significant role in the malignant pleural mesothelioma (MPM) etiology. The differential impact on MPM risk of polymorphic alleles of glutathione- S-transferases ( GSTs) and manganese superoxide dismutase ( MnSOD/SOD2) genes involved in the defence against oxidative damage has been investigated. Ninety cases of MPM and 395 controls were genotyped using the arrayed-primer extension technique. Logistic regression analysis was applied to assess the predictive role of single nucleotide polymorphisms (SNPs) potentially involved in MPM carcinogenesis after adjustment for potential confounders. An increased risk of MPM was found in subjects bearing a GSTM1 null allele (OR = 1.69, 95% CI = 1.04-2.74; p = 0.034), and in those with the Ala/Ala genotypes at codon 16 within MnSOD (OR = 3.07, 95% CI = 1.55-6.05; p = 0.001). A stronger effect of MnSOD was observed among patients without a clear exposure to asbestos fibres. No effect was found for GSTA2, GSTA4, GSTM3, GSTP1 and GSTT1 genes. These findings, if replicated, contribute substantial evidence to the hypothesis that oxidative stress and cellular antireactive oxygen species systems are involved in the pathogenesis and in the natural history of MPM. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Objectives: The rarity of familial neuroblastoma (NB) has allowed only a few linkage studies, most of which did not show any evidence of linkage to regions involved in somatic alterations or to genes implicated in other neurocristopathies seldom associated with NB. We screened a highly informative family with recurrent NB by genome-wide linkage analysis aimed at identifying chromosomal regions for NB predisposing genes. Methods: A genome-wide screen was performed using 382 microsatellite markers. Multipoint model-based linkage analysis was carried out under a dominant mode of inheritance for the disease using the ‘affected only’ approach. Results: Our analysis identified two haplotypes co-segregating with the disease on chromosomes 2p and 12p, and yielded maximum lod-score values of 3.01 (p < 0.0001) for markers on both intervals. Conclusions: Evidence of linkage was reported at 16p in North American families, whereas our studies excluded this interval and indicated other loci for disease predisposition, thus confirming the remarkable genetic heterogeneity of NB. These results suggest an oligogenic inheritance in NB involving more loci in genetic determination of the disease. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The hypermethylation of CpG islands within gene promoter regions is an epigenetic phenomenon that is often, but not always, associated with the transcriptional silencing of downstream genes and contributes to carcinogenesis. We have determined the pattern of methylation of several genes involved in distinct biological pathways, including cell proliferation and apoptosis, in neuroblastoma and in the nonmalignant ganglioneuroma. The purpose of this work was to search for epigenetic signatures that could be associated with defined clinical and biological parameters and that, in prospective, could identify specific risk categories among the patients. We have analysed 31 malignant neuroblastoma with or without MYCN amplification and 13 benign ganglioneuroma and we have observed dramatic differences in the methylation pattern of five genes (CASP8, 14.3.3σ, ΔN-p73, RASSF1A and DCR2) between these tumors indicating that this phenomenon is not tissue-specific and can be considered as cancer-dependent. Furthermore, the methylation pattern of 14.3.3σ, RASSF1A and of an intragenic segment of CASP8 was significantly different between MYCN amplified and single copy neuroblastoma suggesting a specific role of epigenetic alterations in aggressive neuroblastoma.Oncogene (2005) 24, 5619–5628. doi:10.1038/sj.onc.1208722; published online 25 July 2005 [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is a childhood malignancy originating from neural crest cells, which seldom occurs in association with other neurocristopathies. Owing to the rarity of familial NB cases, only a few linkage data are available and no mutations in candidate genes have been demonstrated up till now. Germline mutations in a small proportion of NB patients have been recently reported in the paired-like homeobox 2B (PHOX2B) gene, suggesting its role in NB predisposition. On the basis of this indication, we screened three Italian families with recurrence of NB and one family with occurrence of ganglioneuroblastoma and isolated Hirschsprung disease for PHOX2B defects. Our analysis did not show any mutation, excluding PHOX2B as the NB susceptibility gene in the families we analysed. Our findings combined with those derived from other PHOX2B mutation screenings and from genome-wide linkage analysis support a remarkable genetic heterogeneity of NB and suggest an oligogenic model of disease transmission. Furthermore, as PHOX2B mutations were mainly observed in some NB families with multifocal and syndromic NB, features that are missing in the families we have studied, we suggest they represent second-site modifications responsible for a specific phenotype rather than causal mutations of a major locus.Oncogene (2005) 24, 3050-3053. doi:10.1038/sj.onc.1208532 Published online 28 February 2005 [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the histological, immunohistochemical and molecular features of a series of children with neuroblastic tumors (NTs) and opsoclonus-myoclonus syndrome (OMS). Of 1187 children (age 0-15 years) with previously untreated NTs registered between 1979 and 1995, 15 (1.3%) had OMS at presentation. The majority of patients showed favorable biological characteristics, such as lack of amplification of the neuroblastoma-associated avian myelocytomatosis homolog MYCN oncogene and aneuploid nuclear DNA content. Tumor histology was reviewed according to the International Neuroblastoma Pathology Classification. Histology of the 15 cases of NTs with OMS was ganglioneuroblastoma, intermixed, in 10 patients; ganglioneuroma, maturing, in 1; and neuroblastoma in 4. Of 15 tumors, 12 (10 ganglioneuroblastomas, 2 neuroblastomas) showed abundant interstitial or perivascular lymphoid infiltrates, the latter often organized in secondary lymphoid follicles. The three remaining cases had only minimal infiltrates. A review of 91 cases of age- and stage-matched neuroblastic tumors not associated with OMS tested as controls showed that the degree of lymphoid infiltration was significantly lower than that detected in OMS-related tumors. Furthermore, lymphoid follicles were always present in the latter tumors, whereas they were detected only in a few ganglioneuroma, intermixed tumors from the control group. In conclusion, ganglioneuroblastoma, intermixed subtype, lack of MYCN amplification, aneuploid DNA content and presence of lymphoid infiltrates may contribute to favorable prognosis in NTs associated with OMS. [ABSTRACT FROM AUTHOR]

The most frequent genetic alterations described in neuroblastoma (NB) are amplification of MYCN oncogene and deletion of chromosome 1p, although somatic deletions have been demonstrated at other chromosomal intervals. Since loss of heterozygosity (LOH) at distal 4p has been observed in about 20–29% of neuroblastomas, we have evaluated deletions in 41 Italian NB samples by LOH analysis at loci mapping to 4p as follows: pter-D4S2936-D4S412-D4S2957-D4S432-D4S3023-D4S431-cen. Our analysis showed allele losses in eight out of 41 samples (19.5%) and allowed the identification of a smallest region of overlapping deletion (SRO) of 3.0 cM, delimited by D4S412 and D4S3023. Two of these tumors with 4p LOH are from patients belonging to a family with recurrent NB. Interestingly the genotyping of this family revealed an identical haplotype that includes the nonrecombinant loci D4S412, D4S2957 and D4S432 shared by all affected children and demonstrated that this haplotype is retained in the two tumors carrying somatic deletions from patients of this family. Furthermore linkage analysis was performed in two NB families and yielded an overall lod-score of 3.0 in the interval including the haplotype. This provides a confirmatory indication that the region delimited by D4S2936 and D4S3023, which also includes the new defined SRO, may harbor NB predisposing gene/s.Oncogene (2002) 21, 8356–8360. doi:10.1038/sj.onc.1206009 [ABSTRACT FROM AUTHOR]

Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12–24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein.... [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is the most common extra-cranial malignancy in preschool children. To portray the genetic landscape of an overly aggressive NB leading to a rapid clinical progression of the disease, tumor DNA collected pre- and post-treatment has been analyzed. Array comparative genomic hybridization (aCGH), whole-exome sequencing (WES), and pharmacogenetics approaches, respectively, have identified relevant copy number alterations (CNAs), single nucleotide variants (SNVs), and polymorphisms (SNPs) that were then combined into an integrated analysis. Spontaneously formed 3D tumoroids obtained from the recurrent mass have also been characterized. The results prove the power of combining CNAs, SNVs, and SNPs analyses to assess clonal evolution during the disease progression by evidencing multiple clones at disease onset and dynamic genomic alterations during therapy administration. The proposed molecular and cytogenetic integrated analysis empowers the disease follow-up and the prediction of tumor recurrence. [ABSTRACT FROM AUTHOR]

Neuroblastoma is a pediatric cancer, onset with localized as well as metastatic disease. Localized tumors usually show a high content of aneuploid cells. It is suggested that aneuploid cells with numerical copy number variation (CNV) are generated by chromosome instability (CIN). Patients with a localized tumor respond well to the therapy and show a good outcome. On the contrary, patients with a metastatic tumor have worse outcomes and the cells with structural CNV show high levels of CIN. It is proposed that a favorable outcome in patients with localized disease is associated to the grade of CIN. [ABSTRACT FROM AUTHOR]