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1.  Clinical Characteristics and Outcome of Patients with Neuroblastoma Presenting Genomic Amplification of Loci Other than MYCN 
PLoS ONE  2014;9(7):e101990.
Background
Somatically acquired genomic alterations with MYCN amplification (MNA) are key features of neuroblastoma (NB), the most common extra-cranial malignant tumour of childhood. Little is known about the frequency, clinical characteristics and outcome of NBs harbouring genomic amplification(s) distinct from MYCN.
Methods
Genomic profiles of 1100 NBs from French centres studied by array-CGH were re-examined specifically to identify regional amplifications. Patients were included if amplifications distinct from the MYCN locus were seen. A subset of NBs treated at Institut Curie and harbouring MNA as determined by array-CGH without other amplification was also studied. Clinical and histology data were retrospectively collected.
Results
In total, 56 patients were included and categorised into 3 groups. Group 1 (n = 8) presented regional amplification(s) without MNA. Locus 12q13-14 was a recurrent amplified region (4/8 cases). This group was heterogeneous in terms of INSS stages, primary localisations and histology, with atypical clinical features. Group 2 (n = 26) had MNA as well as other regional amplifications. These patients shared clinical features of those of a group of NBs MYCN amplified (Group 3, n = 22). Overall survival for group 1 was better than that of groups 2 and 3 (5 year OS: 87.5%±11% vs 34.9%±7%, log-rank p<0.05).
Conclusion
NBs harbouring regional amplification(s) without MNA are rare and seem to show atypical features in clinical presentation and genomic profile. Further high resolution genetic explorations are justified in this heterogeneous group, especially when considering these alterations as predictive markers for targeted therapy.
doi:10.1371/journal.pone.0101990
PMCID: PMC4094484  PMID: 25013904
2.  Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma 
Oncotarget  2014;5(9):2688-2702.
Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.
PMCID: PMC4058037  PMID: 24811913
Neuroblastoma; ALK; neurogenesis; therapeutic target; RET
3.  SegAnnDB: interactive Web-based genomic segmentation 
Bioinformatics  2014;30(11):1539-1546.
Motivation: DNA copy number profiles characterize regions of chromosome gains, losses and breakpoints in tumor genomes. Although many models have been proposed to detect these alterations, it is not clear which model is appropriate before visual inspection the signal, noise and models for a particular profile.
Results: We propose SegAnnDB, a Web-based computer vision system for genomic segmentation: first, visually inspect the profiles and manually annotate altered regions, then SegAnnDB determines the precise alteration locations using a mathematical model of the data and annotations. SegAnnDB facilitates collaboration between biologists and bioinformaticians, and uses the University of California, Santa Cruz genome browser to visualize copy number alterations alongside known genes.
Availability and implementation: The breakpoints project on INRIA GForge hosts the source code, an Amazon Machine Image can be launched and a demonstration Web site is http://bioviz.rocq.inria.fr.
Contact: toby@sg.cs.titech.ac.jp
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu072
PMCID: PMC4029035  PMID: 24493034
4.  Breakpoint Features of Genomic Rearrangements in Neuroblastoma with Unbalanced Translocations and Chromothripsis 
PLoS ONE  2013;8(8):e72182.
Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.
doi:10.1371/journal.pone.0072182
PMCID: PMC3753337  PMID: 23991058
5.  Learning smoothing models of copy number profiles using breakpoint annotations 
BMC Bioinformatics  2013;14:164.
Background
Many models have been proposed to detect copy number alterations in chromosomal copy number profiles, but it is usually not obvious to decide which is most effective for a given data set. Furthermore, most methods have a smoothing parameter that determines the number of breakpoints and must be chosen using various heuristics.
Results
We present three contributions for copy number profile smoothing model selection. First, we propose to select the model and degree of smoothness that maximizes agreement with visual breakpoint region annotations. Second, we develop cross-validation procedures to estimate the error of the trained models. Third, we apply these methods to compare 17 smoothing models on a new database of 575 annotated neuroblastoma copy number profiles, which we make available as a public benchmark for testing new algorithms.
Conclusions
Whereas previous studies have been qualitative or limited to simulated data, our annotation-guided approach is quantitative and suggests which algorithms are fastest and most accurate in practice on real data. In the neuroblastoma data, the equivalent pelt.n and cghseg.k methods were the best breakpoint detectors, and exhibited reasonable computation times.
doi:10.1186/1471-2105-14-164
PMCID: PMC3712326  PMID: 23697330
6.  ALK germline mutations in patients with neuroblastoma: a rare and weakly penetrant syndrome 
Neuroblastic tumours may occur in a predisposition context. Two main genes are involved: PHOX2B, observed in familial cases and frequently associated with other neurocristopathies (Ondine's and Hirschsprung's disease); and ALK, mostly in familial tumours. We have assessed the frequency of mutations of these two genes in patients with a presumable higher risk of predisposition. We sequenced both genes in 26 perinatal cases (prebirth and <1 month of age, among which 10 were multifocal), 16 multifocal postnatal (>1 month) cases, 3 pairs of affected relatives and 8 patients with multiple malignancies. The whole coding sequences of the two genes were analysed in tumour and/or constitutional DNAs. We found three ALK germline mutations, all in a context of multifocal tumours. Two mutations (T1151R and R1192P) were inherited and shared by several unaffected patients, thus illustrating an incomplete penetrance. Younger age at tumour onset did not seem to offer a relevant selection criterion for ALK analyses. Conversely, multifocal tumours might be the most to benefit from the genetic screening. Finally, no PHOX2B germline mutation was found in this series. In conclusion, ALK deleterious mutations are rare events in patients with a high probability of predisposition. Other predisposing genes remain to be discovered.
doi:10.1038/ejhg.2011.195
PMCID: PMC3283184  PMID: 22071890
ALK; neuroblastoma; predisposition
7.  Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data 
Bioinformatics  2011;28(3):423-425.
Summary: More and more cancer studies use next-generation sequencing (NGS) data to detect various types of genomic variation. However, even when researchers have such data at hand, single-nucleotide polymorphism arrays have been considered necessary to assess copy number alterations and especially loss of heterozygosity (LOH). Here, we present the tool Control-FREEC that enables automatic calculation of copy number and allelic content profiles from NGS data, and consequently predicts regions of genomic alteration such as gains, losses and LOH. Taking as input aligned reads, Control-FREEC constructs copy number and B-allele frequency profiles. The profiles are then normalized, segmented and analyzed in order to assign genotype status (copy number and allelic content) to each genomic region. When a matched normal sample is provided, Control-FREEC discriminates somatic from germline events. Control-FREEC is able to analyze overdiploid tumor samples and samples contaminated by normal cells. Low mappability regions can be excluded from the analysis using provided mappability tracks.
Availability: C++ source code is available at: http://bioinfo.curie.fr/projects/freec/
Contact: freec@curie.fr
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr670
PMCID: PMC3268243  PMID: 22155870
8.  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
9.  High Cyclin E Staining Index in Blastemal, Stromal or Epithelial Cells Is Correlated with Tumor Aggressiveness in Patients with Nephroblastoma 
PLoS ONE  2008;3(5):e2216.
Purpose
Identifying among nephroblastoma those with a high propensity for distant metastases using cell cycle markers: cyclin E as a regulator of progression through the cell cycle and Ki-67 as a tumor proliferation marker, since both are often deregulated in many human malignancies.
Methodology/Principal Findings
A staining index (SI) was obtained by immunohistochemistry using anti-cyclin E and anti-Ki-67 antibodies in paraffin sections of 54 postchemotherapy nephroblastoma including 42 nephroblastoma without metastasis and 12 with metastases. Median cyclin E and Ki-67 SI were 46% and 33% in blastemal cells, 30% and 10% in stromal cells, 37% and 29.5% in epithelial cells. The highest values were found for anaplastic nephroblastoma. A correlation between cyclin E and Ki-67 SI was found for the blastemal component and for the epithelial component. Univariate analysis showed prognostic significance for metastases with cyclin E SI in stromal cells, epithelial cells and blastemal cells (p = 0.03, p = 0.01 and p = 0.002, respectively) as well as with Ki-67 SI in blastema (p<10−4). The most striking data were that both cyclin E SI and blastemal Ki-67 SI discriminated between patients with metastases and patients without metastasis among intermediate-risk nephroblastoma.
Conclusions
Our findings show that a high cyclin E SI in all components of nephroblastoma is correlated with tumor aggressiveness and metastases, and that assessment of its expression may have prognostic value in the categorization of nephroblastoma.
doi:10.1371/journal.pone.0002216
PMCID: PMC2373888  PMID: 18493303

Results 1-9 (9)