PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (25)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
more »
1.  A p53 Drug Response Signature Identifies Prognostic Genes in High-Risk Neuroblastoma 
PLoS ONE  2013;8(11):e79843.
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.
doi:10.1371/journal.pone.0079843
PMCID: PMC3865347  PMID: 24348903
2.  ERBB3 is a marker of a ganglioneuroblastoma/ganglioneuroma-like expression profile in neuroblastic tumours 
Molecular Cancer  2013;12:70.
Background
Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. However, by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. In the current study we characterized these different subgroups in more detail, with a specific focus on the fourth divergent tumour subgroup (r4).
Methods
Expression microarray data from four international studies corresponding to 148 neuroblastic tumour cases were subject to division into four expression subgroups using a previously described 6-gene signature. Differentially expressed genes between groups were identified using Significance Analysis of Microarray (SAM). Next, gene expression network modelling was performed to map signalling pathways and cellular processes representing each subgroup. Findings were validated at the protein level by immunohistochemistry and immunoblot analyses.
Results
We identified several significantly up-regulated genes in the r4 subgroup of which the tyrosine kinase receptor ERBB3 was most prominent (fold change: 132–240). By gene set enrichment analysis (GSEA) the constructed gene network of ERBB3 (n = 38 network partners) was significantly enriched in the r4 subgroup in all four independent data sets. ERBB3 was also positively correlated to the ErbB family members EGFR and ERBB2 in all data sets, and a concurrent overexpression was seen in the r4 subgroup. Further studies of histopathology categories using a fifth data set of 110 neuroblastic tumours, showed a striking similarity between the expression profile of r4 to ganglioneuroblastoma (GNB) and ganglioneuroma (GN) tumours. In contrast, the NB histopathological subtype was dominated by mitotic regulating genes, characterizing unfavourable NB subgroups in particular. The high ErbB3 expression in GN tumour types was verified at the protein level, and showed mainly expression in the mature ganglion cells.
Conclusions
Conclusively, this study demonstrates the importance of performing unsupervised clustering and subtype discovery of data sets prior to analyses to avoid a mixture of tumour subtypes, which may otherwise give distorted results and lead to incorrect conclusions. The current study identifies ERBB3 as a clear-cut marker of a GNB/GN-like expression profile, and we suggest a 7-gene expression signature (including ERBB3) as a complement to histopathology analysis of neuroblastic tumours. Further studies of ErbB3 and other ErbB family members and their role in neuroblastic differentiation and pathogenesis are warranted.
doi:10.1186/1476-4598-12-70
PMCID: PMC3766266  PMID: 23835063
Microarray; Expression; Cancer; Systems biology; Oncology; Network; Reverse engineering; Unsupervised; Clustering; Cell cycle; Spindle assembly; Her-3; HER3; ERBB3; Her-2; HER2; ERBB2; EGFR; ERBB1; BIRC5; Survivin; MYCN; N-myc; ALK; PHOX2B; NTRK1; CCND1
3.  Pharmacological activation of the p53 pathway by nutlin-3 exerts anti-tumoral effects in medulloblastomas 
Neuro-Oncology  2012;14(7):859-869.
Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40%–70%, their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. Here, we analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum and assessed the mutational status of p53 and MDM2 expression in 6 medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas, compared with cerebellum. Four of 6 medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability and induced either cell cycle arrest or apoptosis and expression of the p53 target gene p21 in these 4 cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials.
doi:10.1093/neuonc/nos115
PMCID: PMC3379802  PMID: 22591662
MDM2; medulloblastoma; nutlin-3; p21; wild-type p53
4.  MYCN and HDAC2 cooperate to repress miR-183 signaling in neuroblastoma 
Nucleic Acids Research  2013;41(12):6018-6033.
MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function.
doi:10.1093/nar/gkt346
PMCID: PMC3695529  PMID: 23625969
5.  Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers 
Genome Biology  2012;13(10):R95.
Background
Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.
Results
To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2'-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.
Conclusions
This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.
doi:10.1186/gb-2012-13-10-r95
PMCID: PMC3491423  PMID: 23034519
6.  Cancer Gene Prioritization for Targeted Resequencing Using FitSNP Scores 
PLoS ONE  2012;7(3):e31333.
Background
Although the throughput of next generation sequencing is increasing and at the same time the cost is substantially reduced, for the majority of laboratories whole genome sequencing of large cohorts of cancer samples is still not feasible. In addition, the low number of genomes that are being sequenced is often problematic for the downstream interpretation of the significance of the variants. Targeted resequencing can partially circumvent this problem; by focusing on a limited number of candidate cancer genes to sequence, more samples can be included in the screening, hence resulting in substantial improvement of the statistical power. In this study, a successful strategy for prioritizing candidate genes for targeted resequencing of cancer genomes is presented.
Results
Four prioritization strategies were evaluated on six different cancer types: genes were ranked using these strategies, and the positive predictive value (PPV) or mutation rate within the top-ranked genes was compared to the baseline mutation rate in each tumor type. Successful strategies generate gene lists in which the top is enriched for known mutated genes, as evidenced by an increase in PPV. A clear example of such an improvement is seen in colon cancer, where the PPV is increased by 2.3 fold compared to the baseline level when 100 top fitSNP genes are sequenced.
Conclusions
A gene prioritization strategy based on the fitSNP scores appears to be most successful in identifying mutated cancer genes across different tumor entities, with variance of gene expression levels as a good second best.
doi:10.1371/journal.pone.0031333
PMCID: PMC3291573  PMID: 22396732
7.  N-Cadherin in Neuroblastoma Disease: Expression and Clinical Significance 
PLoS ONE  2012;7(2):e31206.
One of the first and most important steps in the metastatic cascade is the loss of cell-cell and cell-matrix interactions. N-cadherin, a crucial mediator of homotypic and heterotypic cell-cell interactions, might play a central role in the metastasis of neuroblastoma (NB), a solid tumor of neuroectodermal origin. Using Reverse Transcription Quantitative PCR (RT-qPCR), Western blot, immunocytochemistry and Tissue MicroArrays (TMA) we demonstrate the expression of N-cadherin in neuroblastoma tumors and cell lines. All neuroblastic tumors (n = 356) and cell lines (n = 10) expressed various levels of the adhesion protein. The N-cadherin mRNA expression was significantly lower in tumor samples from patients suffering metastatic disease. Treatment of NB cell lines with the N-cadherin blocking peptide ADH-1 (Exherin, Adherex Technologies Inc.), strongly inhibited tumor cell proliferation in vitro by inducing apoptosis. Our results suggest that N-cadherin signaling may play a role in neuroblastoma disease, marking involvement of metastasis and determining neuroblastoma cell viability.
doi:10.1371/journal.pone.0031206
PMCID: PMC3280274  PMID: 22355346
8.  Chromosomal and miRNA Expression Patterns Reveal Biologically Distinct Subgroups of 11q− Neuroblastoma 
Purpose
The purpose of this study was to further define the biology of the 11q− neuroblastoma tumor subgroup by the integration of aCGH with miRNA expression profiling data to determine if improved patient stratification is possible.
Experimental Design
A set of primary neuroblastoma (n=160) which was broadly representative of all genetic subtypes was analyzed by aCGH and for the expression of 430 miRNAs. A 15 miRNA expression signature previously demonstrated to be predictive of clinical outcome was used to analyze an independent cohort of 11q− tumors (n=37).
Results
Loss of 4p and gain of 7q occurred at a significantly higher frequency in the 11q−tumors, further defining the genetic characteristics of this subtype. The 11q− tumors could be split into two subgroups using a miRNA expression survival signature which differed significantly in both clinical outcome and the overall frequency of large scale genomic imbalances, with the poor survival subgroup having significantly more imbalances. MiRNAs from the expression signature which were up-regulated in unfavorable tumors were predicted to target down-regulated genes from a published mRNA expression classifier of clinical outcome at a higher than expected frequency, indicating the miRNAs might contribute to the regulation of genes within the signature.
Conclusion
We demonstrate that two distinct biological subtypes of neuroblastoma with loss of 11q occur which differ in their miRNA expression profiles, frequency of segmental imbalances and clinical outcome. A miRNA expression signature, combined with an analysis of segmental imbalances, provides greater prediction of EFS and OS outcomes than 11q status by itself, improving patient stratification.
doi:10.1158/1078-0432.CCR-09-3215
PMCID: PMC2880207  PMID: 20406844
aCGH; MYCN; neuroblastoma; miRNA
9.  A 6-gene signature identifies four molecular subgroups of neuroblastoma 
Background
There are currently three postulated genomic subtypes of the childhood tumour neuroblastoma (NB); Type 1, Type 2A, and Type 2B. The most aggressive forms of NB are characterized by amplification of the oncogene MYCN (MNA) and low expression of the favourable marker NTRK1. Recently, mutations or high expression of the familial predisposition gene Anaplastic Lymphoma Kinase (ALK) was associated to unfavourable biology of sporadic NB. Also, various other genes have been linked to NB pathogenesis.
Results
The present study explores subgroup discrimination by gene expression profiling using three published microarray studies on NB (47 samples). Four distinct clusters were identified by Principal Components Analysis (PCA) in two separate data sets, which could be verified by an unsupervised hierarchical clustering in a third independent data set (101 NB samples) using a set of 74 discriminative genes. The expression signature of six NB-associated genes ALK, BIRC5, CCND1, MYCN, NTRK1, and PHOX2B, significantly discriminated the four clusters (p < 0.05, one-way ANOVA test). PCA clusters p1, p2, and p3 were found to correspond well to the postulated subtypes 1, 2A, and 2B, respectively. Remarkably, a fourth novel cluster was detected in all three independent data sets. This cluster comprised mainly 11q-deleted MNA-negative tumours with low expression of ALK, BIRC5, and PHOX2B, and was significantly associated with higher tumour stage, poor outcome and poor survival compared to the Type 1-corresponding favourable group (INSS stage 4 and/or dead of disease, p < 0.05, Fisher's exact test).
Conclusions
Based on expression profiling we have identified four molecular subgroups of neuroblastoma, which can be distinguished by a 6-gene signature. The fourth subgroup has not been described elsewhere, and efforts are currently made to further investigate this group's specific characteristics.
doi:10.1186/1475-2867-11-9
PMCID: PMC3095533  PMID: 21492432
10.  Outcome Prediction of Children with Neuroblastoma using a Multigene Expression Signature, a Retrospective SIOPEN/COG/GPOH Study 
The lancet oncology  2009;10(7):663-671.
BACKGROUND
More accurate prognostic assessment of patients with neuroblastoma is required to improve the choice of risk-related therapy. The aim of this study is to develop and validate a gene expression signature for improved outcome prediction.
METHODS
Fifty-nine genes were carefully selected based on an innovative data-mining strategy and profiled in the largest neuroblastoma patient series (n=579) to date using RT-qPCR starting from only 20 ng of RNA. A multigene expression signature was built using 30 training samples, tested on 313 test samples and subsequently validated in a blind study on an independent set of 236 additional tumours.
FINDINGS
The signature accurately classifies patients with respect to overall and progression-free survival (p<0·0001). The signature has a performance, sensitivity, and specificity of 85·4% (95%CI: 77·7–93·2), 84·4% (95%CI: 66·5–94·1), and 86·5% (95%CI: 81·1–90·6), respectively to predict patient outcome. Multivariate analysis indicates that the signature is a significant independent predictor after controlling for currently used riskfactors. Patients with high molecular risk have a higher risk to die from disease and for relapse/progression than patients with low molecular risk (odds ratio of 19·32 (95%CI: 6·50–57·43) and 3·96 (95%CI: 1·97–7·97) for OS and PFS, respectively). Patients with increased risk for adverse outcome can also be identified within the current treatment groups demonstrating the potential of this signature for improved clinical management. These results were confirmed in the validation study in which the signature was also independently statistically significant in a model adjusted for MYCN status, age, INSS stage, ploidy, INPC grade of differentiation, and MKI. The high patient/gene ratio (579/59) underlies the observed statistical power and robustness.
INTERPRETATION
A 59-gene expression signature predicts outcome of neuroblastoma patients with high accuracy. The signature is an independent risk predictor, identifying patients with increased risk in the current clinical risk groups. The applied method and signature is suitable for routine lab testing and ready for evaluation in prospective studies.
FUNDING
The Belgian Foundation Against Cancer, found of public interest (project SCIE2006-25), the Children Cancer Fund Ghent, the Belgian Society of Paediatric Haematology and Oncology, the Belgian Kid’s Fund and the Fondation Nuovo-Soldati (JV), the Fund for Scientific Research Flanders (KDP, JH), the Fund for Scientific Research Flanders (grant number: G•0198•08), the Institute for the Promotion of Innovation by Science and Technology in Flanders, Strategisch basisonderzoek (IWT-SBO 60848), the Fondation Fournier Majoie pour l’Innovation, the Instituto Carlos III,RD 06/0020/0102 Spain, the Italian Neuroblastoma Foundation, the European Community under the FP6 (project: STREP: EET-pipeline, number: 037260), and the Belgian program of Interuniversity Poles of Attraction, initiated by the Belgian State, Prime Minister's Office, Science Policy Programming.
doi:10.1016/S1470-2045(09)70154-8
PMCID: PMC3045079  PMID: 19515614
11.  Measurable impact of RNA quality on gene expression results from quantitative PCR 
Nucleic Acids Research  2011;39(9):e63.
Compromised RNA quality is suggested to lead to unreliable results in gene expression studies. Therefore, assessment of RNA integrity and purity is deemed essential prior to including samples in the analytical pipeline. This may be of particular importance when diagnostic, prognostic or therapeutic conclusions depend on such analyses. In this study, the comparative value of six RNA quality parameters was determined using a large panel of 740 primary tumour samples for which real-time quantitative PCR gene expression results were available. The tested parameters comprise of microfluidic capillary electrophoresis based 18S/28S rRNA ratio and RNA Quality Index value, HPRT1 5′–3′ difference in quantification cycle (Cq) and HPRT1 3′ Cq value based on a 5′/3′ ratio mRNA integrity assay, the Cq value of expressed Alu repeat sequences and a normalization factor based on the mean expression level of four reference genes. Upon establishment of an innovative analytical framework to assess impact of RNA quality, we observed a measurable impact of RNA quality on the variation of the reference genes, on the significance of differential expression of prognostic marker genes between two cancer patient risk groups, and on risk classification performance using a multigene signature. This study forms the basis for further rational assessment of reverse transcription quantitative PCR based results in relation to RNA quality.
doi:10.1093/nar/gkr065
PMCID: PMC3089491  PMID: 21317187
12.  The emerging molecular pathogenesis of neuroblastoma: implications for improved risk assessment and targeted therapy 
Genome Medicine  2009;1(7):74.
Neuroblastoma is one of the most common solid tumors of childhood, arising from immature sympathetic nervous system cells. The clinical course of patients with neuroblastoma is highly variable, ranging from spontaneous regression to widespread metastatic disease. Although the outcome for children with cancer has improved considerably during the past decades, the prognosis of children with aggressive neuroblastoma remains dismal. The clinical heterogeneity of neuroblastoma mirrors the biological and genetic heterogeneity of these tumors. Ploidy and MYCN amplification have been used as genetic markers for risk stratification and therapeutic decision making, and, more recently, gene expression profiling and genome-wide DNA copy number analysis have come into the picture as sensitive and specific tools for assessing prognosis. The applica tion of new genetic tools also led to the discovery of an important familial neuroblastoma cancer gene, ALK, which is mutated in approximately 8% of sporadic tumors, and genome-wide association studies have unveiled loci with risk alleles for neuroblastoma development. For some of the genomic regions that are deleted in some neuroblastomas, on 1p, 3p and 11q, candidate tumor suppressor genes have been identified. In addition, evidence has emerged for the contribution of epigenetic disturbances in neuroblastoma oncogenesis. As in other cancer entities, altered microRNA expression is also being recognized as an important player in neuroblastoma. The recent successes in unraveling the genetic basis of neuroblastoma are now opening opportunities for development of targeted therapies.
doi:10.1186/gm74
PMCID: PMC2717400  PMID: 19638189
13.  RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts 
BMC Research Notes  2009;2:235.
Background
The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR.
Findings
The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification.
Conclusion
Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.
doi:10.1186/1756-0500-2-235
PMCID: PMC2789097  PMID: 19930725
14.  External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data 
Nucleic Acids Research  2009;37(21):e138.
The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.
doi:10.1093/nar/gkp721
PMCID: PMC2790878  PMID: 19734345
15.  CADM1 is a strong neuroblastoma candidate gene that maps within a 3.72 Mb critical region of loss on 11q23 
BMC Cancer  2008;8:173.
Background
Recurrent loss of part of the long arm of chromosome 11 is a well established hallmark of a subtype of aggressive neuroblastomas. Despite intensive mapping efforts to localize the culprit 11q tumour suppressor gene, this search has been unsuccessful thus far as no sufficiently small critical region could be delineated for selection of candidate genes.
Methods
To refine the critical region of 11q loss, the chromosome 11 status of 100 primary neuroblastoma tumours and 29 cell lines was analyzed using a BAC array containing a chromosome 11 tiling path. For the genes mapping within our refined region of loss, meta-analysis on published neuroblastoma mRNA gene expression datasets was performed for candidate gene selection. The DNA methylation status of the resulting candidate gene was determined using re-expression experiments by treatment of neuroblastoma cells with the demethylating agent 5-aza-2'-deoxycytidine and bisulphite sequencing.
Results
Two small critical regions of loss within 11q23 at chromosomal band 11q23.1-q23.2 (1.79 Mb) and 11q23.2-q23.3 (3.72 Mb) were identified. In a first step towards further selection of candidate neuroblastoma tumour suppressor genes, we performed a meta-analysis on published expression profiles of 692 neuroblastoma tumours. Integration of the resulting candidate gene list with expression data of neuroblastoma progenitor cells pinpointed CADM1 as a compelling candidate gene. Meta-analysis indicated that CADM1 expression has prognostic significance and differential expression for the gene was noted in unfavourable neuroblastoma versus normal neuroblasts. Methylation analysis provided no evidence for a two-hit mechanism in 11q deleted cell lines.
Conclusion
Our study puts CADM1 forward as a strong candidate neuroblastoma suppressor gene. Further functional studies are warranted to elucidate the role of CADM1 in neuroblastoma development and to investigate the possibility of CADM1 haploinsufficiency in neuroblastoma.
doi:10.1186/1471-2407-8-173
PMCID: PMC2442116  PMID: 18559103
16.  Positional gene enrichment analysis of gene sets for high-resolution identification of overrepresented chromosomal regions 
Nucleic Acids Research  2008;36(7):e43.
The search for feature enrichment is a widely used method to characterize a set of genes. While several tools have been designed for nominal features such as Gene Ontology annotations or KEGG Pathways, very little has been proposed to tackle numerical features such as the chromosomal positions of genes. For instance, microarray studies typically generate gene lists that are differentially expressed in the sample subgroups under investigation, and when studying diseases caused by genome alterations, it is of great interest to delineate the chromosomal regions that are significantly enriched in these lists. In this article, we present a positional gene enrichment analysis method (PGE) for the identification of chromosomal regions that are significantly enriched in a given set of genes. The strength of our method relies on an original query optimization approach that allows to virtually consider all the possible chromosomal regions for enrichment, and on the multiple testing correction which discriminates truly enriched regions versus those that can occur by chance. We have developed a Web tool implementing this method applied to the human genome (http://www.esat.kuleuven.be/~bioiuser/pge). We validated PGE on published lists of differentially expressed genes. These analyses showed significant overrepresentation of known aberrant chromosomal regions.
doi:10.1093/nar/gkn114
PMCID: PMC2367735  PMID: 18346969
17.  Correction: Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes 
Genome Biology  2007;8(1):401.
A correction to Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes by K De Preter, J Vandesompele, P Heimann, N Yigit, S Beckman, A Schramm, A Eggert, RL Stallings, Y Benoit, M Renard, A De Paepe, G Laureys, S Påhlman and F Speleman. Genome Biology 2006 7:R84
doi:10.1186/gb-2007-8-1-401
PMCID: PMC1839119
18.  Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes 
Genome Biology  2006;7(9):R84.
Transcriptome profiling of neuroblasts and neuroblastoma tumor cells provides strong support for a neuroblast origin of neuroblastoma and highlights new candidate neuroblastoma genes
Background
Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands.
Results
Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis.
Conclusion
This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.
doi:10.1186/gb-2006-7-9-r84
PMCID: PMC1794547  PMID: 16989664
19.  Positional and functional mapping of a neuroblastoma differentiation gene on chromosome 11 
BMC Genomics  2005;6:97.
Background
Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11.
Results
In a first step, we performed high-resolution arrayCGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene.
Conclusion
Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.
doi:10.1186/1471-2164-6-97
PMCID: PMC1185534  PMID: 16000168
20.  arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays 
BMC Bioinformatics  2005;6:124.
Background
The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment.
Results
We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser.
Conclusion
ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at .
doi:10.1186/1471-2105-6-124
PMCID: PMC1173083  PMID: 15910681
21.  No evidence for involvement of SDHD in neuroblastoma pathogenesis 
BMC Cancer  2004;4:55.
Background
Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma.
Methods
SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria.
Results
Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype.
Conclusions
Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis.
doi:10.1186/1471-2407-4-55
PMCID: PMC517501  PMID: 15331017
22.  Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons 
BMC Genomics  2004;5:11.
Background
Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes.
Results
As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.
Conclusions
The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.
doi:10.1186/1471-2164-5-11
PMCID: PMC365025  PMID: 15018647
Amplification; Overexpression; Oncogene; SSH; Array CGH; Neuroblastoma
23.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes 
Genome Biology  2002;3(7):research0034.1-research0034.11.
Using real-time reverse transcription PCR ten housekeeping genes from different abundance and functional classes in various human tissues were evaluated. The conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested.
Background
Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.
Results
We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.
Conclusions
The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
PMCID: PMC126239  PMID: 12184808
24.  Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes 
PLoS ONE  2013;8(1):e52321.
Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.
doi:10.1371/journal.pone.0052321
PMCID: PMC3537730  PMID: 23308108
25.  Multiplex Amplicon Quantification (MAQ), a fast and efficient method for the simultaneous detection of copy number alterations in neuroblastoma 
BMC Genomics  2010;11:298.
Background
Cancer genomes display characteristic patterns of chromosomal imbalances, often with diagnostic and prognostic relevance. Therefore assays for genome-wide copy number screening and simultaneous detection of copy number alterations in specific chromosomal regions are of increasing importance in the diagnostic work-up of tumors.
Results
We tested the performance of Multiplex Amplicon Quantification, a newly developed low-cost, closed-tube and high-throughput PCR-based technique for detection of copy number alterations in regions with prognostic relevance for neuroblastoma. Comparison with array CGH and the established Multiplex Ligation-dependent Probe Amplification method on 52 neuroblastoma tumors showed that Multiplex Amplicon Quantification can reliably detect the important genomic aberrations.
Conclusion
Multiplex Amplicon Quantification is a low-cost and high-throughput PCR-based technique that can reliably detect copy number alterations in regions with prognostic relevance for neuroblastoma.
doi:10.1186/1471-2164-11-298
PMCID: PMC2879279  PMID: 20459859

Results 1-25 (25)