Outcome In Neuroblastoma Research Articles

Classification of neuroblastoma patients by published gene-expression markers reveals a low sensitivity for unfavorable courses of MYCN non-amplified disease

Currently, Pubmed lists 385 marker genes for neuroblastoma outcome. Using a customized neuroblastoma-microarray, we evaluated the prognostic impact of the gene-expression pattern of 349 of these candidates (90.6%) in 127 neuroblastoma patients with divergent outcome. By significance analysis of microarrays (SAM) and both uncorrected and Bonferroni-corrected ANOVA, 166/349 (47.5%), 218/349 (62.5%) and 128/349 (36.4%) candidates showed significant differential expression between patients with contrasting outcome. By Prediction Analysis for Microarrays (PAM), a 38-gene-classifier was derived from all markers, which classified patients outcome with an overall accuracy of 78.5%. However, patients with unfavorable outcome of MYCN non-amplified disease were largely misclassified (accuracy: 35%), suggesting that these courses are not identified by current marker genes.

Elevated multidrug resistance-associated protein 4 (MRP4) expression in localized prostate cancer—A potential androgen regulated protein

20022 Background: MRP4 is an ATP-binding cassette transporter and amphipathic anion efflux pump which transports prostaglandins, nucleoside analogues, glutamate and phosphate analogues. High MRP4 expression is prognostic of poor outcome in neuroblastoma and also correlates with MYCN amplification, suggesting regulation by this oncogene. Although MRP4 is known to be expressed in normal prostate epithelium, its expression in prostate cancer (PC) is undefined. This study aimed to define the pattern of expression of MRP4 in normal and malignant prostate tissue and assess the association with androgen exposure. Methods: 84 radical prostatectomy specimens from patients with clinically localized PC (22 neoadjuvant androgen ablation therapy, 62 no neoadjuvant treatment), 42 specimens of hyperplasia adjacent to PC and 16 cases of advanced PC were assessed for MRP4 expression using in situ hybridisation and immunohistochemistry. PC cell lines were assessed by immunoblotting. Results: There were significantly higher levels of MRP4 mRNA and protein expression in localized PC compared to hyperplasia (p=0.006). Conversely, MRP4 protein levels were significantly decreased in PCs treated with neoadjuvant androgen ablation therapy compared to cancers exposed to normal testosterone levels (p < 0.0001). There was also a trend towards decreased MRP4 expression in advanced PCs. Furthermore, immunoblotting revealed that MRP4 protein was more highly expressed in androgen-dependent (LNCaP) compared to androgen-independent (PC3/DU145) cell lines. In addition, in a panel of 14 normal human tissues only kidney and prostate tissue expressed MRP4 protein suggesting limited expression of MRP4 in human tissues. Discussion: Elevated MRP4 expression is found in malignant compared to benign prostate tissue while lower MRP4 expression is seen after androgen ablation suggesting that MRP4 may be an androgen-regulated gene. In addition, there is relatively little expression of MRP4 in normal tissues. These data suggest that MRP4 is important in the progression to PC and that it may be a potential therapeutic target. No significant financial relationships to disclose.

Chromosome 1p and 11q Deletions and Outcome in Neuroblastoma

Chromosome 1p and 11q Deletions and Outcome in Neuroblastoma

Favorable outcome of triploid neuroblastomas: a contribution to the special oncogenesis of neuroblastoma

There is a well-known association between patient outcome and tumor ploidy in neuroblastoma. To date, however, most clinical trials have not used this parameter for therapy stratification. Using conventional cytogenetics and fluorescence in situ hybridization (FISH), we investigated 36 tumors in terms of ploidy and chromosome 1 copy number (polysomy). In addition, interphase FISH for polysomy was performed on a second cohort of 440 neuroblastomas, together with the status of 1p, MYCN, and 11q. The main goals were as follows: (1) to assess the reliability of FISH to determine ploidy; (2) to illustrate associations between somy 1 and clinical/biologic factors; and (3) to investigate the role of somy 1 for predicting outcome. The comparison between karyotyping and FISH in the smaller cohort revealed 86% consistency between ploidy and polysomy (31/36). According to FISH, trisomic tumors in the second cohort showed structural chromosomal aberrations less frequently compared to di-/tetrasomic tumors (15 vs. 60%, P < 0.001). The portion of trisomic neuroblastomas was higher in stages 1, 2, and 4S versus stages 3 and 4 (55 vs. 24%, P < 0.001) and in children 18 months or younger versus those older than 18 months (55 vs. 19%, P < 0.001). Prognosis was significantly better for trisomic tumors versus di-/tetrasomic in the whole cohort [event-free (EFS) and overall survival (OS), P < 0.001]. In the subgroup without abnormalities of other molecular markers, EFS of trisomic neuroblastomas was better ( P = 0.048), but was most likely due to an unequal stage distribution. In further subgroups, in terms of age and stage, significance between the somy groups was not reached, neither for EFS nor OS. The multivariate analyses including age, stage, chromosomal markers, and somy 1 confirmed the lack of independent prognostic power for the copy number of chromosome 1. This study demonstrates the following: (1) FISH is a practical alternative to other more labor-intensive techniques for determining ploidy; (2) trisomic tumors correlate with younger age at diagnosis, localized stage, and the lack of structural alterations; and (3) polysomy is not an independent prognostic marker. The sharp decline of trisomic tumors after the age of 18 months supports the idea of different genetic tumor entities.

Association of High-Level MRP1 Expression With Poor Clinical Outcome in a Large Prospective Study of Primary Neuroblastoma

We have previously shown in a retrospective study that expression of the multidrug transporter gene MRP1 (ABCC1) is associated with outcome in neuroblastoma. We have now undertaken a prospective analysis to examine the independent prognostic significance of MRP1 expression in a large cohort of primary untreated neuroblastomas. Two hundred nine diagnostic neuroblastoma samples from patients prospectively enrolled onto the Pediatric Oncology Group biology protocol 9047 were analyzed for expression of the MRP1, MDR1, MYCN, and TRKA genes using real-time polymerase chain reaction. Expression levels were correlated with established prognostic indicators and disease outcome. MRP1 expression was detected in all tumors analyzed, and levels were significantly higher in tumors with versus without MYCN amplification (P < .0001). High levels of MRP1 were highly predictive of both event-free survival (EFS; P < .001) and overall survival (OS; P < .001). High-level MYCN and low-level TRKA were also predictive of poor outcome. MDR1 expression demonstrated no prognostic significance. After adjustment for the effect of statistically significant prognostic indicators in multivariate models, MRP1 expression retained significant prognostic value for both EFS (hazard ratio = 3.0; P = .0011) and OS (hazard ratio = 2.5; P = .0095), whereas MYCN amplification did not have prognostic significance. The results of this prospective study confirm our earlier findings and support a clinically relevant role for MRP1 gene expression in neuroblastoma. These findings have implications for the biology, prognosis, and treatment of this disease and provide evidence that MRP1 is a bone fide molecular target for reversing chemotherapy resistance in aggressive drug-refractory neuroblastoma.

TheMYCNEnigma: Significance ofMYCNExpression in Neuroblastoma

MYCN amplification strongly predicts adverse outcome of neuroblastoma. However, the significance of MYCN expression in the clinical and biological behavior of neuroblastoma has been unclear. To address this question, we first examined the expression of MYCN in combination with TrkA (a favorable prognostic indicator of neuroblastoma) in 91 primary neuroblastoma by quantitative reverse transcription-PCR and investigated the relationship among patient survival, MYCN, and TrkA expressions. Three subsets of neuroblastoma were defined based on MYCN and TrkA expression. Neuroblastoma expressing the highest level of MYCN but little TrkA were MYCN-amplified cases, which had a 5-year survival of 9.3%. Interestingly, MYCN and TrkA expression showed a linear correlation (r = 0.5664, P < 0.00005) in neuroblastoma lacking MYCN amplification, and the 5-year survival of neuroblastoma patients with low MYCN and low TrkA expressions was 63.7%, whereas those with high expression of both had a 5-year survival of 88.1% (P < 0.00005). This nonlinear distribution of disease outcome relative to MYCN expression in neuroblastoma explains why MYCN expression is not predictive of neuroblastoma disease outcome by dichotomous division of the neuroblastoma cohort. However, high-level MYCN expression is associated with favorable outcome in neuroblastoma lacking MYCN amplification. Furthermore, forced expression of MYCN significantly suppresses growth of neuroblastoma cells lacking MYCN amplification by inducing apoptosis and enhancing favorable neuroblastoma gene expression. Collectively, these data suggest that high-level MYCN expression in neuroblastoma lacking MYCN amplification results in a benign phenotype. Thus, the high MYCN expression confers the opposite biological consequence in neuroblastoma, depending on whether or not MYCN is amplified.

The Influence of Biologic Factors on the Surgical Decision in Advanced Neuroblastoma

Molecular markers greatly affect the outcome of neuroblastoma. This study evaluated the influence of Trk-A and myelocytomatosis viral-related oncogene, neuroblastoma-derived (MYCN) on the role of surgery in advanced neuroblastoma. Ten stage 3 and 35 stage 4 neuroblastoma patients were included. Tumor resection was classified into gross total resection (GTR) and incomplete resection. Patients were classified into three biological risk groups according to Trk-A expression and myelocytomatosis viral-related oncogene, neuroblastoma-derived (MYCN) status in tumor tissues studied by immunohistochemistry and fluorescence in situ hybridization, respectively: low risk (positive Trk-A and normal MYCN), intermediate risk (negative Trk-A and normal MYCN), and high risk (positive or negative Trk-A and MYCN amplification). The effect of tumor resection on prognosis was studied and stratified according to the risk grouping. GTR was achieved in 21 patients (46.7%) with a higher complication rate (33% vs. 8% in the incomplete resection group, P = .036). GTR was easier to achieve in low-risk tumors than in intermediate- or high-risk tumors (12 of 13, 4 of 17, and 5 of 15, respectively; P < .001). GTR predicted a favorable prognosis for intermediate-risk patients (P = .037; log-rank test), but not for low- or high-risk patients because of the overall favorable and poor prognosis, respectively. GTR carries a potentially higher possibility of complication. Although GTR can be achieved easily in low-risk neuroblastoma patients with a favorable prognosis, surgeons should do their best to achieve GTR for intermediate-risk patients to improve outcome. Nevertheless, sacrificing vital organs to achieve GTR for high-risk patients is not justified.

Chromosome 1p and 11q Deletions and Outcome in Neuroblastoma

Neuroblastoma is a childhood cancer with considerable morbidity and mortality. Tumor-derived biomarkers may improve risk stratification. We screened 915 samples of neuroblastoma for loss of heterozygosity (LOH) at chromosome bands 1p36 and 11q23. Additional analyses identified a subgroup of cases of 11q23 LOH with unbalanced 11q LOH (unb11q LOH; defined as loss of 11q with retention of 11p). The associations of LOH with relapse and survival were determined. LOH at 1p36 was identified in 209 of 898 tumors (23 percent) and LOH at 11q23 in 307 of 913 (34 percent). Unb11q LOH was found in 151 of 307 tumors with 11q23 LOH (17 percent of the total cohort). There was a strong association of 1p36 LOH, 11q23 LOH, and unb11q LOH with most high-risk disease features (P<0.001). LOH at 1p36 was associated with amplification of the MYCN oncogene (P<0.001), but 11q23 LOH and unb11q LOH were not (P<0.001 and P=0.002, respectively). Cases with unb11q LOH were associated with three-year event-free and overall survival rates (+/-SE) of 50+/-5 percent and 66+/-5 percent, respectively, as compared with 74+/-2 percent and 83+/-2 percent among cases without unb11q LOH (P<0.001 for both comparisons). In a multivariate model, unb11q LOH was independently associated with decreased event-free survival (P=0.009) in the entire cohort, and both 1p36 LOH and unb11q LOH were independently associated with decreased progression-free survival in the subgroup of patients with features of low-risk and intermediate-risk disease (P=0.002 and P=0.02, respectively). Unb11q LOH and 1p36 LOH are independently associated with a worse outcome in patients with neuroblastoma.

Prediction of clinical outcome and biological characterization of neuroblastoma by expression profiling

Neuroblastoma is a common childhood tumor comprising cases with rapid disease progression as well as spontaneous regression. Although numerous prognostic factors have been identified, risk evaluation in individual patients remains difficult. To define a reliable prognostic predictor and gene signatures characteristic of biological subgroups, we performed mRNA expression profiling of 68 neuroblastomas of all stages. Expression data were analysed using support vector machines (SVM-rbf), prediction analysis of microarrays (PAM), k-nearest neighbors (k-NN) algorithms and multiple decision trees. SVM-rbf performed best of all methods, and predicted recurrence of neuroblastoma with an accuracy of 85% (sensitivity 77%, specificity 94%). PAM identified a classifier of 39 genes reliably predicting outcome with an accuracy of 80%. In comparison, conventional risk stratification based on stage, age and MYCN-status only reached a predictive accuracy of 64%. Kaplan-Meier analysis using the PAM classifier indicated a 5-year survival of 20 versus 78% for patients with unfavorably versus favorably predicted neuroblastomas, respectively (P = 0.0001). Significance analysis of microarrays (SAM) identified additional genes differentially expressed among subgroups. MYCN-amplification and high expression of NTRK1/TrkA demonstrated a strong association with specific gene expression patterns. Our data suggest that microarray-derived data in addition to traditional clinical factors will be useful for risk assessment and defining biological properties of neuroblastoma.

Chromosome 11 Abnormality Linked to Poor Outcome in Neuroblastoma

Chromosome 11 Abnormality Linked to Poor Outcome in Neuroblastoma

Chromosome arm 11q deletion predicts for neuroblastoma outcome: A Children’s Oncology Group study

6 Background: Chromosome arm 11q loss of heterozygosity (LOH) is a frequent occurrence in neuroblastoma. Previous studies have shown an association between 11q LOH and high-risk disease. However, these studies lacked the statistical power to conclusively analyze the prognostic impact of 11q LOH in multivariable analyses after adjusting for the currently used prognostic factors. Methods: We screened 917 primary neuroblastomas for chromosome 11 LOH with a panel of 4–8 microsatellite markers. Further mapping with up to 52 markers was performed on selected samples. The only inclusion criteria were availability of a constitutional DNA sample and outcome data. The sample set was representative of the overall neuroblastoma population. The median follow-up was 2.7 years. Results: LOH at chromosome arm 11q and unbalanced 11q LOH (loss of 11q material with retention or gain of 11p material) were present in 302 (33%) and 148 (16%) cases, respectively. The pattern of LOH for each tumor sample was consistent with the presence of a single region of deletion; all but two deletions involved 11q23. The majority of tumors with 11q LOH did not have MYCN amplification. Despite this, both 11q LOH and unbalanced 11q LOH were associated with high-risk disease (p<0.0001). Patients whose tumors showed unbalanced 11q LOH had 3-year EFS and OS of 50%±6% and 65%±5%, compared to 74%±2% (p<0.0001) and 83%±2% (p<0.0001) in those cases that did not (log-rank test). In a Cox proportional hazards model for multivariable analysis, unbalanced 11q LOH was independently prognostic for EFS after adjusting for stage 4, MYCN amplification, and Shimada histology (p=0.0068). Conclusion: Unbalanced 11q LOH is associated with aggressive neuroblastoma behavior and is independently prognostic for decreased EFS. Unbalanced 11q LOH status could be especially useful in determining whether a patient with locoregional neuroblastoma without MYCN amplification would benefit from more adjuvant chemotherapy. No significant financial relationships to disclose.

Identification of Dynamic Proteome Changes Upon Ligand Activation of Trk-Receptors Using Two-dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

Association of Epigenetic Inactivation of RASSF1A with Poor Outcome in Human Neuroblastoma

To investigate the prevalence and potential clinical significance of epigenetic aberrations in neuroblastoma (NB). The methylation status of 11 genes that are frequently epigenetically inactivated in adult cancers was assayed in 13 NB cell lines. The prevalence of RASSF1A and TSP-1 methylation was also analyzed in 56 NBs and 5 ganglioneuromas by methylation-specific PCR. Associations between the methylation status of RASSF1A and TSP-1 and patient age, tumor stage, tumor MYCN status, and patient survival were evaluated. Epigenetic changes were detected in all 13 NB cell lines, although the pattern of gene methylation varied. The putative tumor suppressor gene RASSF1A was methylated in all 13 cell lines, and TSP-1 and CASP8 were methylated in 11 of 13 cell lines. Epigenetic changes of DAPK and FAS were detected in only small numbers of cell lines, whereas none of the cell lines had methylation of p16, p21, p73, RAR-beta2, SPARC, or TIMP-3. RASSF1A was also methylated in 70% of the primary NB tumors tested, and TSP-1 methylation was detected in 55% of the tumors. RASSF1A methylation was significantly associated with age >1 year (P < 0.01), high-risk disease (P < 0.016), and poor survival (P < 0.001). In contrast, no association between TSP-1 methylation and prognostic factors or survival was observed. Our results suggest that epigenetic inactivation of RASSF1A may contribute to the clinically aggressive phenotype of high-risk NB.

Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas

Expression of neurotrophin receptors of the tyrosine kinase receptor (Trk) family is an important prognostic factor in solid tumors including neuroblastoma. High expression of TrkA (NTRK1) is associated with a favorable biology and outcome of neuroblastoma, whereas TrkB (NTRK2) is expressed on aggressive neuroblastomas with unfavorable outcome. To gain new insights into the global gene expression program resulting in these divergent biological phenotypes, we stably expressed either TrkA or TrkB in the human SH-SY5Y neuroblastoma cell line. Gene expression profiles were obtained from parental cells and transfectants activated by their ligands in a time course over 24 h using oligonucleotide microarrays. Basal activation of Trk receptors in the absence of exogenous ligand was sufficient to induce broad and divergent genetic changes. Global gene regulation following external ligand stimulation was surprisingly similar in SY5Y-TrkA and SY5Y-TrkB cells except for the differential expression of distinct novel target genes. Consistent with their divergent biological phenotype, SY5Y-TrkA cells were characterized by upregulation of proapoptotic genes and angiogenesis inhibitors, whereas SY5Y-TrkB cells demonstrated upregulation of genes involved in invasion or therapy resistance. We suggest that the transcriptional program of neuroblastoma cells is modulated by Trk-receptor expression and basal activation rather than by ligand-induced activation. Fine-tuning of the malignant phenotype may be achieved by additional ligand stimulation with subsequent activation of a few specific genes.

Hepatocyte growth factor/c-Met signaling promotes the progression of experimental human neuroblastomas.

Neuroblastoma is the most frequent solid childhood malignancy. Despite aggressive therapy, mortality is high due to rapid tumor progression to advanced stages. The molecules and mechanisms underlying poor prognosis are not well understood. Here, we report that cultured human neuroblastoma cells express the hepatocyte growth factor (HGF) and its receptor c-Met. Binding of HGF to c-Met triggers receptor autophosphorylation, indicating functional relevance of this interaction. HGF activates several downstream effectors of c-Met such as the mitogen-activated protein kinases extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 and phospholipase C-gamma, whereas signal transducer and activator of transcription 3 is constitutively activated in neuroblastoma cells expressing c-Met. In addition, HGF is able to stimulate expression and proteolytic activity of matrix metalloproteinase-2 and tissue-type plasminogen activator in neuroblastoma cells, thereby promoting degradation of extracellular matrix components. We show that HGF stimulates invasion of neuroblastoma cells in vitro and in vivo, and it promotes the formation of angiogenic neuroblastomas in vivo. These processes can be blocked by specific inhibitors of the mitogen-activated protein kinase cascade, by inhibitors of phospholipase C-gamma, and also by the expression of a dominant negative signal transducer and activator of transcription 3 mutant. Our data provide the first evidence that the HGF/c-Met pathway is essential for invasiveness and malignant progression of human neuroblastomas. They further suggest that specific inhibitors of this pathway may be suitable as therapeutic agents to improve clinical outcome of neuroblastomas.

The Brn-3b Transcription Factor Regulates the Growth, Behavior, and Invasiveness of Human Neuroblastoma Cells in Vitro and in Vivo

Neuroblastomas are the second most common solid tumor in children but the molecular mechanisms underlying the initiation and progression of this disease are poorly understood. We previously showed that the Brn-3b transcription factor is highly expressed in actively proliferating neuroblastoma cells but is significantly decreased when these cells are induced to differentiate. In this study, we analyzed the effects of manipulating Brn-3b levels in the human neuroblastoma cell line, IMR-32 and showed that constitutive overexpression of Brn-3b consistently increased cellular growth and proliferation in monolayer as well as in an anchorage-independent manner compared with controls whereas stably decreasing Brn-3b can reduce the rate of growth of these cells. Cells with high Brn-3b also fail to respond to growth inhibitory retinoic acid, as they continue to proliferate. Moreover, Brn-3b levels significantly modified tumor growth in vivo with elevated Brn-3b resulting in faster tumor growth in xenograft models whereas decreasing Brn-3b resulted in slower growth compared with controls. Interestingly, elevated Brn-3b levels also enhances the invasive capacity of these neuroblastoma cells with significantly larger numbers of migrating cells observed in overexpressing clones compared with controls. Because invasion and metastasis influence morbidity and mortality in neuroblastoma and so significantly affect the course and outcome of neuroblastomas, this finding is very important. Our results therefore suggest that Brn-3b transcription factor contributes to proliferation of neuroblastoma cells in vivo and in vitro but may also influence progression and/or invasion during tumorigenesis. It is possible that decreasing Brn-3b levels may reverse some effects on growth and proliferation of these cells.

Prediction of clinical outcome and biological characterization of neuroblastoma by expression profiling

Prediction of clinical outcome and biological characterization of neuroblastoma by expression profiling

Arginine deiminase and other antiangiogenic agents inhibit unfavorable neuroblastoma growth: potentiation by irradiation.

In spite of aggressive therapy, children suffering from neuroblastoma have a poor prognosis. Therapeutic failure is most often observed in neuroblastomas with unfavorable features, including amplification/over-expression of the N-myc oncogene, rapid growth, effective angiogenesis and/or the tendency to metastasize. Here, we have used cultured human neuroblastoma cells with such features and we have examined whether antiangiogenic agents alone or in combination with tumor irradiation inhibit their angiogenesis and growth in vivo. We report that antiangiogenic agents (arginine deiminase, SU5416 and DC101) inhibit in vivo growth of neuroblastomas with unfavorable properties and that these effects are potentiated by simultaneous irradiation. Combination of either agent with irradiation leads to a reduction in the absolute number of tumor vessels and of perfused tumor vessels. Combination of arginine deiminase or DC101 with irradiation does not increase tumor hypoxia. Our data demonstrate for the first time that arginine deiminase suppresses the growth of unfavorable experimental neuroblastomas and that this effect is potentiated by irradiation. We suggest that antiangiogenesis alone or in combination with established therapeutic regimen may improve the outcome of unfavorable neuroblastomas in a clinical setting.

The impact of surgical radicality on outcome in childhood neuroblastoma

But: Une amelioration des resultats dans le neuroblastome (NB) a ete realisee durant ces dernieres annees et essentiellement par une intensification du traitement pour les formes evoluees de neuroblastome. La chirurgie, malgre tout, n'a pas beaucoup contribue a ce progres et il reste encore des controverses concernant la meilleur approche des neuroblastomes a haut risque. Malgre tout, nous cherchons des criteres pour definir la strategie de la resection tumorale dans NB. Methodes: Nous avons etudie retrospectivement les dossiers de 2250 patients traites pour NB dans l'etude cooperative allemande NB79 - NB97 (1979-1999). L'âge des patients, le stade tumorale, le statut N-MYC, les possibilites chirurgicales, le caractere complet de la resection, les complications chirurgicales et le devenir ont ete analyses. Resultats: 1148 patients avaient une forme NB localisee (stade 1-3), 878 un stade 4 et 225 un stade 4 S. 2112 patients ont subi une chirurgie. La resection tumorale comme resultat apres une intervention premiere ou secondaire etait complete chez 1403 (66.4%), incomplete chez 449 (21.3%) et seulement une biopsie etait realisee chez 260 (12.3%). Une resection complete etait realisee le plus souvent dans les formes localisee de NB (73.5%), moins souvent dans les stade 4 (59.2%), et dans les stades 4S (54.5%). Les complications sont survenues dans 19.2% de l'ensemble des operations, independamment du moment de la resection, de l'âge des patients, du stade de la tumeur, du site de la tumeur et du statut N-MYC. Pour 1787 patients de l'etude NB79 - NB90, la probabilite d'un suivi sans complications a 5 ans (EFS) pouvait etre analyse et correle au caractere radical de la resection. Dans les formes localisees des patients de plus de 1 an, il y avait une difference significative pour l'EFS a 5 ans entre la resection complete, incomplete et l'absence de resection (p < 0.001), alors qu'il n'y avait pas de difference pour les enfants de moins de 1 an. Dans la premiere periode de l'etude (NB79 -NB90), les patients avec un stade 4 avaient une EFS a 5 ans significativement meilleur apres excision complete de la tumeur primitive. En contraste, les differences n'etaient pas confirmees durant la derniere periode (NB90) avec une chimiotherapie intensifiee. Le moment de la chirurgie n'influence pas le pronostic. Pour les enfants avec un stade 4 S, il n'y a pas de difference de devenir apres resection complete ou incomplete de la tumeur primitive. Le statut N-MYC ne modifie pas l'interet de la resection tumorale pour le devenir des formes localisees de NB. Au contraire des stades 4 sans amplifications de N-MYC, le stade 4 avec amplification de N-MYC a un meilleur devenir si la tumeur primitive peut etre completement ou incompletement resequee, en comparaison a une biopsie seulement. Conclusion: Les resections radicales de la tumeur avec le risque chirurgical de complications est justifie chez les enfants de plus de 1 an avec une forme localisee du neuroblastome. Dans le stade 4 de NB, la tumeur primitive doit etre resequee durant un traite? ment multimodal sans risque de complications. Seulement dans les formes stade 4 amplifiant N-MYC, la resection complete ou incomplete de la tumeur primitive ameliore le resultat. Le stade 4 S avec N-MYC non-amplifie sont sujets a des regressions spontanees, une resection apres chimiotherapie peut-etre indiquee en cas de regression non suffisante ou d'augmentation de la tumeur primitive.

The MYCN oncoprotein as a drug development target

The transcription factor and proto-oncogene MYCN is reviewed as a potential specific target for cancer therapy. Amplification of MYCN is frequently found in a number of advanced-stage tumours, including neuroblastoma (25%), small cell lung cancers (7%), alveolar rhabdomyosarcoma and retinoblastoma. It is associated with rapid tumour progression and poor outcome in human neuroblastoma. MYCN is a member of the myc family of proto-oncogenes which encode nuclear proteins that form heterodimers with MAX protein through their conserved HLHZip domains. The MYC/MAX complexes transactivate a number of MYC-target genes in a sequence-specific manner. MYC-MAX interaction is essential for MYC-induced cell cycle progression, cellular transformation, and transcriptional activation. A causal link between the transformed phenotype and MYCN has been established by a range of in vitro and in vivo studies, including a transgenic model of neuroblastoma in which MYCN overexpression is targeted to neuronal tissue by the use of a tyrosine hydroxylase promoter. Downregulation of MYCN expression either by antisense treatment targeted against MYCN mRNA or by retinoids has been shown to decrease proliferation and/or induce neuronal differentiation of neuroblastoma cells. Inhibition of MYC-MAX dimerisation by small-molecule antagonists has recently been shown to interfere with MYC-induced transformation of chick embryo fibroblasts, indicating that functional inhibitors of the MYC family of oncoproteins have potential as therapeutic agents. Finally, we describe the development and validation of a functional MYCN reporter gene assay using neuroblastoma cells (NGP) which have been stably transfected with a luciferase gene construct under control of the ornithine decarboxylase gene promoter. This assay has been used for a pilot screen of 2800 compounds from the Cancer Research-UK collection, identifying five compounds showing a consistent significant reduction of MYCN-dependent luciferase activity (>50%) in repeated screens. This cell-based, MYCN reporter gene assay will be scaled up for high throughput screens of compound libraries and will aid in the future development of specific therapeutic strategies in neuroblastoma and other tumours in which MYCN amplification has been implicated.