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.
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.
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).
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.
Nephroblastoma (Wilms' tumor; WT) is the most common renal tumor of childhood. To date, several genetic abnormalities predisposing to WT have been identified in rare overgrowth syndromes. Among them, abnormal methylation of the 11p15 region, GPC3 and DIS3L2 mutations, which are responsible for Beckwith–Wiedemann, Simpson–Golabi–Behmel and Perlman syndromes, respectively. However, the underlying cause of WT remains unknown in the majority of cases. We report three unrelated patients who presented with WT in addition to a constitutional 9q22.3 microdeletion and dysmorphic/overgrowth syndrome. The size of the deletions was variable (ie, from 1.7 to 8.9 Mb) but invariably encompassed the PTCH1 gene. Subsequently, we identified a somatic PTCH1 nonsense mutation in the renal tumor of one patient. In addition, by array comparative genomic hybridization method, we analyzed the DNA extracted from the blood samples of nine patients with overgrowth syndrome and WT, but did not identify any deleterious chromosomal imbalances in these patients. These findings strongly suggest that patients with constitutional 9q22.3 microdeletion have an increased risk of WT, and that PTCH1 have a role in the pathogenesis of nephroblastomas.
CNV; PTCH1; FANCC nephroblastoma; overgrowth; Wilms' tumor; Perlman syndrome
The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification.
Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC-1 parental cells in nude mice generated various tumor types, such as NB, osteo/chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.
ALK; neuroblastoma; Myc; tumorigenesis; differentiation
Pure invasive micropapillary carcinoma (IMPC) is a special type of breast carcinoma characterised by clusters of cells presenting polarity abnormalities. The biological alterations underlying this pattern remain unknown.
Pangenomic analysis (n = 39), TP53 (n = 43) and PIK3CA (n = 41) sequencing in a series of IMPCs were performed. A subset of cases was also analysed with whole-exome sequencing (n = 4) and RNA sequencing (n = 6). Copy number variation profiles were compared with those of oestrogen receptors and grade-matched invasive ductal carcinomas (IDCs) of no special type.
Unsupervised analysis of genomic data distinguished two IMPC subsets: one (Sawtooth/8/16) exhibited a significant increase in 16p gains (71%), and the other (Firestorm/Amplifier) was characterised by a high frequency of 8q (35%), 17q (20% to 46%) and 20q (23% to 30%) amplifications and 17p loss (74%). TP53 mutations (10%) were more frequently identified in the amplifier subset, and PIK3CA mutations (4%) were detected in both subsets. Compared to IDC, IMPC exhibited specific loss of the 6q16-q22 region (45%), which is associated with downregulation of FOXO3 and SEC63 gene expression. SEC63 and FOXO3 missense mutations were identified in one case each (2%). Whole-exome sequencing combined with RNA sequencing of IMPC allowed us to identify somatic mutations in genes involved in polarity, DNAH9 and FMN2 (8% and 2%, respectively) or ciliogenesis, BBS12 and BBS9 (2% each) or genes coding for endoplasmic reticulum protein, HSP90B1 and SPTLC3 (2% each) and cytoskeleton, UBR4 and PTPN21 (2% each), regardless of the genomic subset. The intracellular biological function of the mutated genes identified by gene ontology analysis suggests a driving role in the clinicopathological characteristics of IMPC.
In our comprehensive molecular analysis of IMPC, we identified numerous genomic alterations without any recurrent fusion genes. Recurrent somatic mutations of genes participating in cellular polarity and shape suggest that they, together with other biological alterations (such as epigenetic modifications and stromal alterations), could contribute to the morphological pattern of IMPC. Though none of the individual abnormalities demonstrated specificity for IMPC, whether their combination in IMPC may have a cumulative effect that drives the abnormal polarity of IMPC needs to be examined further with in vitro experiments.
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.
Neuroblastoma; ALK; neurogenesis; therapeutic target; RET
The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.
ALK; brainstem; neonatal lethality; plethysmography; feeding difficulties
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.
Supplementary data are available at Bioinformatics online.
The accurate prognosis definition to tailor treatment for early luminal invasive breast carcinoma patients remains challenging.
Materials and Methods
Two hundred fourteen early luminal breast carcinomas were genotyped with single nucleotide polymorphisms (SNPs) array to determine the number of chromosomal breakpoints as a marker of genomic instability. Proliferation was assessed by KI67 (immunohistochemistry) and genomic grade index (transcriptomic analysis). IHC3 (IHC4 score for HER2 negative tumors) was also determined.
In the training set (109 cases), the optimal cut-off was 34 breakpoints with a specificity of 0.94 and a sensitivity of 0.57 (Area under the curve (AUC): 0.81[0.71; 0.91]). In the validation set (105 cases), the outcome of patients with > 34 breakpoints (11 events / 22 patients) was poorer (logrank test p < 0.001; Relative Risk (RR): 3.7 [1.73; 7.92]), than that of patients with < 34 breakpoints (19 events / 83 patients).Whereas genomic grade and KI67 had a significant prognostic value in univariate analysis in contrast to IHC3 that failed to have a statistical significant prognostic value in this series, the number of breakpoints remained the only significant parameter predictive of outcome (RR: 3.47, Confidence Interval (CI [1.29; 9.31], p = 0.014)) in multivariate analysis .
Genomic instability, defined herein as a high number of chromosomal breakpoints, in early stage luminal breast carcinoma is a stronger prognostic marker than proliferation.
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.
Ewing sarcoma is the second most frequent pediatric bone tumor. In most of the patients, a chromosomal translocation leads to the expression of the EWS-FLI1 chimeric transcription factor that is the major oncogene in this pathology. Relative genetic simplicity of Ewing sarcoma makes it particularly attractive for studying cancer in a systemic manner. Silencing EWS-FLI1 induces cell cycle alteration and ultimately leads to apoptosis, but the exact molecular mechanisms underlying this phenotype are unclear. In this study, a network linking EWS-FLI1 to cell cycle and apoptosis phenotypes was constructed through an original method of network reconstruction. Transcriptome time-series after EWS-FLI1 silencing were used to identify core modulated genes by an original scoring method based on fitting expression profile dynamics curves. Literature data mining was then used to connect these modulated genes into a network. The validity of a subpart of this network was assessed by siRNA/RT-QPCR experiments on four additional Ewing cell lines and confirmed most of the links. Based on the network and the transcriptome data, CUL1 was identified as a new potential target of EWS-FLI1. Altogether, using an original methodology of data integration, we provide the first version of EWS-FLI1 network model of cell cycle and apoptosis regulation.
Medulloblastoma, the most common malignant pediatric brain tumour, is currently treated with non-specific cytotoxic therapies including surgery, whole brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, prior attempts to identify targets for therapy have been underpowered due to small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup enriched. The most common region of focal copy number gain is a tandem duplication of the Parkinson’s disease gene SNCAIP, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1 that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGFβ signaling in Group 3, and NF-κB signaling in Group 4 suggest future avenues for rational, targeted therapy.
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.
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.
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.
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.
ALK; neuroblastoma; predisposition
Pediatric medulloblastoma is considered a highly heterogeneous disease and a new strategy of risk stratification to optimize therapeutic outcomes is required. We aimed to investigate a new risk-stratification approach based on expression profiles of medulloblastoma cohorts. We analyzed gene expression profiles of 30 primary medulloblastomas and detected strong evidence that poor survival outcome was significantly associated with mRNA expression profiles of 17p loss. However, it was not supported in independent cohorts from previously published data (n = 100). We speculated that this discrepancy might come from complex conditions of two important prognostic determinants: loss of tumor suppressors (chromosome 17p) and high expression of oncogenes c-myc (MYCC) or N-myc (MYCN). When patients were stratified into 5 or 7 subgroups based on simultaneous consideration of these 2 factors while defining the Wnt group as independent, obviously different survival expectancies were detected between the subgroups. For instance, predicted 5-year survival probabilities ranged from 19% to 81% in the 5 subgroups. We also found that age became a significant prognostic marker after adjusting for 17p, MYCC, and MYCN status. Diminished survival in age <3 years was more substantial in subgroups with high expression of MYCC, MYCN, or 17p loss but not in other subgroups, indicating that poor survival outcome might be synergistically affected by these 3 factors. Here we suggest a more tailored subgrouping system based on expression profiles of chromosome 17p, MYCC, and MYCN, which could provide the basis for a novel risk-stratification strategy in pediatric medulloblastoma.
chromosome 17p; medulloblastoma; MYC; MYCN; prognosis; Wnt
Social parasitism is an important selective pressure for social insect species. It is particularly the case for the hosts of dulotic (so called slave-making) ants, which pillage the brood of host colonies to increase the worker force of their own colony. Such raids can have an important impact on the fitness of the host nest. An arms race which can lead to geographic variation in host defenses is thus expected between hosts and parasites. In this study we tested whether the presence of a social parasite (the dulotic ant Myrmoxenus ravouxi) within an ant community correlated with a specific behavioral defense strategy of local host or non-host populations of Temnothorax ants. Social recognition often leads to more or less pronounced agonistic interactions between non-nestmates ants. Here, we monitored agonistic behaviors to assess whether ants discriminate social parasites from other ants. It is now well-known that ants essentially rely on cuticular hydrocarbons to discriminate nestmates from aliens. If host species have evolved a specific recognition mechanism for their parasite, we hypothesize that the differences in behavioral responses would not be fully explained simply by quantitative dissimilarity in cuticular hydrocarbon profiles, but should also involve a qualitative response due to the detection of particular compounds. We scaled the behavioral results according to the quantitative chemical distance between host and parasite colonies to test this hypothesis.
Cuticular hydrocarbon profiles were distinct between species, but host species did not show a clearly higher aggression rate towards the parasite than toward non-parasite intruders, unless the degree of response was scaled by the chemical distance between intruders and recipient colonies. By doing so, we show that workers of the host and of a non-host species in the parasitized site displayed more agonistic behaviors (bites and ejections) towards parasite than toward non-parasite intruders.
We used two different analyses of our behavioral data (standardized with the chemical distance between colonies or not) to test our hypothesis. Standardized data show behavioral differences which could indicate qualitative and specific parasite recognition. We finally stress the importance of considering the whole set of potentially interacting species to understand the coevolution between social parasites and their hosts.
Coevolution; Formicidae; Social recognition; Social parasitism; Temnothorax
Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers of response to targeted agents. To uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines, which represent much of the tissue-type and genetic diversity of human cancers, with 130 drugs under clinical and preclinical investigation. In aggregate, we found mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing’s sarcoma cells harboring the EWS-FLI1 gene translocation to PARP inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.
High Content Screening (HCS) platforms allow screening living cells under a wide range of experimental conditions and give access to a whole panel of cellular responses to a specific treatment. The outcome is a series of cell population images. Within these images, the heterogeneity of cellular response to the same treatment leads to a whole range of observed values for the recorded cellular features. Consequently, it is difficult to compare and interpret experiments. Moreover, the definition of phenotypic classes at a cell population level remains an open question, although this would ease experiments analyses. In the present work, we tackle these two questions. The input of the method is a series of cell population images for which segmentation and cellular phenotype classification has already been performed. We propose a probabilistic model to represent and later compare cell populations. The model is able to fully exploit the HCS-specific information: “dependence structure of population descriptors” and “within-population variability”. The experiments we carried out illustrate how our model accounts for this specific information, as well as the fact that the model benefits from considering them. We underline that these features allow richer HCS data analysis than simpler methods based on single cellular feature values averaged over each well. We validate an HCS data analysis method based on control experiments. It accounts for HCS specificities that were not taken into account by previous methods but have a sound biological meaning. Biological validation of previously unknown outputs of the method constitutes a future line of work.
MicroRNAs (miRNAs) have emerged as fundamental regulators that silence gene expression at the post-transcriptional and translational levels. The identification of their targets is a major challenge to elucidate the regulated biological processes. The overall effect of miRNA is reflected on target mRNA expression, suggesting the design of new investigative methods based on high-throughput experimental data such as miRNA and transcriptome profiles. We propose a novel statistical measure of non-linear dependence between miRNA and mRNA expression, in order to infer miRNA-target interactions. This approach, which we name antagonism pattern detection, is based on the statistical recognition of a triangular-shaped pattern in miRNA-target expression profiles. This pattern is observed in miRNA-target expression measurements since their simultaneously elevated expression is statistically under-represented in the case of miRNA silencing effect. The proposed method enables miRNA target prediction to strongly rely on cellular context and physiological conditions reflected by expression data. The procedure has been assessed on synthetic datasets and tested on a set of real positive controls. Then it has been applied to analyze expression data from Ewing’s sarcoma patients. The antagonism relationship is evaluated as a good indicator of real miRNA-target biological interaction. The predicted targets are consistently enriched for miRNA binding site motifs in their 3′UTR. Moreover, we reveal sets of predicted targets for each miRNA sharing important biological function. The procedure allows us to infer crucial miRNA regulators and their potential targets in Ewing’s sarcoma disease. It can be considered as a valid statistical approach to discover new insights in the miRNA regulatory mechanisms.
Medulloblastomas are heterogeneous tumors that collectively represent the most common malignant brain tumor in children. To understand the molecular characteristics underlying their heterogeneity and to identify whether such characteristics represent risk factors for patients with this disease, we performed an integrated genomic analysis of a large series of primary tumors.
Patients and Methods
We profiled the mRNA transcriptome of 194 medulloblastomas and performed high-density single nucleotide polymorphism array and miRNA analysis on 115 and 98 of these, respectively. Non-negative matrix factorization–based clustering of mRNA expression data was used to identify molecular subgroups of medulloblastoma; DNA copy number, miRNA profiles, and clinical outcomes were analyzed for each. We additionally validated our findings in three previously published independent medulloblastoma data sets.
Identified are six molecular subgroups of medulloblastoma, each with a unique combination of numerical and structural chromosomal aberrations that globally influence mRNA and miRNA expression. We reveal the relative contribution of each subgroup to clinical outcome as a whole and show that a previously unidentified molecular subgroup, characterized genetically by c-MYC copy number gains and transcriptionally by enrichment of photoreceptor pathways and increased miR-183∼96∼182 expression, is associated with significantly lower rates of event-free and overall survivals.
Our results detail the complex genomic heterogeneity of medulloblastomas and identify a previously unrecognized molecular subgroup with poor clinical outcome for which more effective therapeutic strategies should be developed.
Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.
Medulloblastoma is the most common malignant brain tumor in childhood. Molecular studies from several groups around the world demonstrated that medulloblastoma is not one disease but comprises a collection of distinct molecular subgroups. However, all these studies reported on different numbers of subgroups. The current consensus is that there are only four core subgroups, which should be termed WNT, SHH, Group 3 and Group 4. Based on this, we performed a meta-analysis of all molecular and clinical data of 550 medulloblastomas brought together from seven independent studies. All cases were analyzed by gene expression profiling and for most cases SNP or array-CGH data were available. Data are presented for all medulloblastomas together and for each subgroup separately. For validation purposes, we compared the results of this meta-analysis with another large medulloblastoma cohort (n = 402) for which subgroup information was obtained by immunohistochemistry. Results from both cohorts are highly similar and show how distinct the molecular subtypes are with respect to their transcriptome, DNA copy-number aberrations, demographics, and survival. Results from these analyses will form the basis for prospective multi-center studies and will have an impact on how the different subgroups of medulloblastoma will be treated in the future.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-012-0958-8) contains supplementary material, which is available to authorized users.
Medulloblastoma; Pediatric brain tumor; Subgroups; Meta-analysis
Identification of new therapeutic agents for breast cancer (BC) requires preclinical models that reproduce the molecular characteristics of their respective clinical tumors. In this work, we analyzed the genomic and gene expression profiles of human BC xenografts and the corresponding patient tumors.
Eighteen BC xenografts were obtained by grafting tumor fragments from patients into Swiss nude mice. Molecular characterization of patient tumors and xenografts was performed by DNA copy number analysis and gene expression analysis using Affymetrix Microarrays.
Comparison analysis showed that 14/18 pairs of tumors shared more than 56% of copy number alterations (CNA). Unsupervised hierarchical clustering analysis showed that 16/18 pairs segregated together, confirming the similarity between tumor pairs. Analysis of recurrent CNA changes between patient tumors and xenografts showed losses in 176 chromosomal regions and gains in 202 chromosomal regions. Gene expression profile analysis showed that less than 5% of genes had recurrent variations between patient tumors and their respective xenografts; these genes largely corresponded to human stromal compartment genes. Finally, analysis of different passages of the same tumor showed that sequential mouse-to-mouse tumor grafts did not affect genomic rearrangements or gene expression profiles, suggesting genetic stability of these models over time.
This panel of human BC xenografts maintains the overall genomic and gene expression profile of the corresponding patient tumors and remains stable throughout sequential in vivo generations. The observed genomic profile and gene expression differences appear to be due to the loss of human stromal genes. These xenografts, therefore, represent a validated model for preclinical investigation of new therapeutic agents.
Breast cancer; xenograft; genomic and expression profiles
The European Network for Cancer Research in Children and Adolescents (ENCCA) provides an interaction platform for stakeholders in research and care of children with cancer. Among ENCCA objectives is the establishment of biology-based prioritization mechanisms for the selection of innovative targets, drugs, and prognostic markers for validation in clinical trials. Specifically for sarcomas, there is a burning need for novel treatment options, since current chemotherapeutic treatment protocols have met their limits. This is most obvious for metastatic Ewing sarcoma (ES), where long term survival rates are still below 20%. Despite significant progress in our understanding of ES biology, clinical translation of promising laboratory results has not yet taken place due to fragmentation of research and lack of an institutionalized discussion forum. To fill this gap, ENCCA assembled 30 European expert scientists and five North American opinion leaders in December 2011 to exchange thoughts and discuss the state of the art in ES research and latest results from the bench, and to propose biological studies and novel promising therapeutics for the upcoming European EWING2008 and EWING2012 clinical trials.
Ewing sarcoma; animal models; sarcomagenesis; genomics; epigenetics; biomarkers; drug screen; prognosis
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/
Supplementary information: Supplementary data are available at Bioinformatics online.
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.
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.
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.
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.
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.