Neuroblastoma is a malignancy of the developing sympathetic nervous system that is often lethal when relapse occurs. We here used whole-exome sequencing, mRNA expression profiling, array CGH and DNA ...methylation analysis to characterize 16 paired samples at diagnosis and relapse from individuals with neuroblastoma. The mutational burden significantly increased in relapsing tumors, accompanied by altered mutational signatures and reduced subclonal heterogeneity. Global allele frequencies at relapse indicated clonal mutation selection during disease progression. Promoter methylation patterns were consistent over disease course and were patient specific. Recurrent alterations at relapse included mutations in the putative CHD5 neuroblastoma tumor suppressor, chromosome 9p losses, DOCK8 mutations, inactivating mutations in PTPN14 and a relapse-specific activity pattern for the PTPN14 target YAP. Recurrent new mutations in HRAS, KRAS and genes mediating cell-cell interaction in 13 of 16 relapse tumors indicate disturbances in signaling pathways mediating mesenchymal transition. Our data shed light on genetic alteration frequency, identity and evolution in neuroblastoma.
To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease.
Two hundred fifty-one neuroblastoma specimens were analyzed using a customized ...oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifier's predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States.
The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival EFS 0.86 +/- 0.03 favorable; n = 115 v 0.52 +/- 0.07 unfavorable; n = 59 and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P < .0001) and separated risk groups of current neuroblastoma trials into subgroups with divergent outcome (NB2004: low-risk 3-year EFS 0.86 +/- 0.04 v 0.25 +/- 0.15, P < .0001; intermediate-risk 1.00 v 0.57 +/- 0.19, P = .018; high-risk 0.81 +/- 0.10 v 0.56 +/- 0.08, P = .06). In a multivariate Cox regression model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 95% CI, 2.544 to 8.893).
Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.
To optimize neuroblastoma treatment stratification, we aimed at developing a novel risk estimation system by integrating gene expression-based classification and established prognostic markers.
Gene ...expression profiles were generated from 709 neuroblastoma specimens using customized 4 × 44 K microarrays. Classification models were built using 75 tumors with contrasting courses of disease. Validation was performed in an independent test set (n = 634) by Kaplan-Meier estimates and Cox regression analyses.
The best-performing classifier predicted patient outcome with an accuracy of 0.95 (sensitivity, 0.93; specificity, 0.97) in the validation cohort. The highest potential clinical value of this predictor was observed for current low-risk patients 5-year event-free survival (EFS), 0.84 ± 0.02 vs. 0.29 ± 0.10; 5-year overall survival (OS), 0.99 ± 0.01 vs. 0.76 ± 0.11; both P < 0.001 and intermediate-risk patients (5-year EFS, 0.88 ± 0.06 vs. 0.41 ± 0.10; 5-year OS, 1.0 vs. 0.70 ± 0.09; both P < 0.001). In multivariate Cox regression models for low-risk/intermediate-risk patients, the classifier outperformed risk assessment of the current German trial NB2004 EFS: hazard ratio (HR), 5.07; 95% confidence interval (CI), 3.20-8.02; OS: HR, 25.54; 95% CI, 8.40-77.66; both P < 0.001. On the basis of these findings, we propose to integrate the classifier into a revised risk stratification system for low-risk/intermediate-risk patients. According to this system, we identified novel subgroups with poor outcome (5-year EFS, 0.19 ± 0.08; 5-year OS, 0.59 ± 0.1), for whom we propose intensified treatment, and with beneficial outcome (5-year EFS, 0.87 ± 0.05; 5-year OS, 1.0), who may benefit from treatment de-escalation.
Combination of gene expression-based classification and established prognostic markers improves risk estimation of patients with low-risk/intermediate-risk neuroblastoma. We propose to implement our revised treatment stratification system in a prospective clinical trial.
BACKGROUND: Current risk stratification systems for neuroblastoma patients consider clinical, histopathological, and genetic variables, and additional prognostic markers have been proposed in recent ...years. We here sought to select highly informative covariates in a multistep strategy based on consecutive Cox regression models, resulting in a risk score that integrates hazard ratios of prognostic variables. METHODS: A cohort of 695 neuroblastoma patients was divided into a discovery set (n=75) for multigene predictor generation, a training set (n=411) for risk score development, and a validation set (n=209). Relevant prognostic variables were identified by stepwise multivariable L1-penalized least absolute shrinkage and selection operator (LASSO) Cox regression, followed by backward selection in multivariable Cox regression, and then integrated into a novel risk score. RESULTS: The variables stage, age, MYCN status, and two multigene predictors, NB-th24 and NB-th44, were selected as independent prognostic markers by LASSO Cox regression analysis. Following backward selection, only the multigene predictors were retained in the final model. Integration of these classifiers in a risk scoring system distinguished three patient subgroups that differed substantially in their outcome. The scoring system discriminated patients with diverging outcome in the validation cohort (5-year event-free survival, 84.9±3.4 vs 63.6±14.5 vs 31.0±5.4; P<.001), and its prognostic value was validated by multivariable analysis. CONCLUSION: We here propose a translational strategy for developing risk assessment systems based on hazard ratios of relevant prognostic variables. Our final neuroblastoma risk score comprised two multigene predictors only, supporting the notion that molecular properties of the tumor cells strongly impact clinical courses of neuroblastoma patients.
Genomic alterations of the anaplastic lymphoma kinase (ALK) gene have been postulated to contribute to neuroblastoma pathogenesis. This study aimed to determine the interrelation of ALK mutations, ...ALK expression levels, and clinical phenotype in primary neuroblastoma.
The genomic ALK status and global gene expression patterns were examined in 263 primary neuroblastomas. Allele-specific ALK expression was determined by cDNA cloning and sequencing. Associations of genomic ALK alterations and ALK expression levels with clinical phenotypes and transcriptomic profiles were compared.
Nonsynonymous point mutations of ALK were detected in 21 of 263 neuroblastomas (8%). Tumors with ALK mutations exhibited about 2-fold elevated median ALK mRNA levels in comparison with tumors with wild-type (WT) ALK. Unexpectedly, the WT allele was preferentially expressed in 12 of 21 mutated tumors. Whereas survival of patients with ALK mutated tumors was significantly worse as compared with the entire cohort of WT ALK patients, it was similarly poor in patients with WT ALK tumors in which ALK expression was as high as in ALK mutated neuroblastomas. Global gene expression patterns of tumors with ALK mutations or with high-level WT ALK expression were highly similar, and suggested that ALK may be involved in cellular proliferation in primary neuroblastoma.
Primary neuroblastomas with mutated ALK exhibit high ALK expression levels and strongly resemble neuroblastomas with elevated WT ALK expression levels in both their clinical and molecular phenotypes. These data suggest that high levels of mutated and WT ALK mediate similar molecular functions that may contribute to a malignant phenotype in primary neuroblastoma.
Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM ...is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. Whole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15). Tumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations. We here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients.
To evaluate the impact of a predefined gene expression-based classifier for clinical risk estimation and cytotoxic treatment decision making in neuroblastoma patients.
Gene expression profiles of 440 ...internationally collected neuroblastoma specimens were investigated by microarray analysis, 125 of which were examined prospectively. Patients were classified as either favorable or unfavorable by a 144-gene prediction analysis for microarrays (PAM) classifier established previously on a separate set of 77 patients. PAM classification results were compared with those of current prognostic markers and risk estimation strategies.
The PAM classifier reliably distinguished patients with contrasting clinical courses (favorable n = 249 and unfavorable n = 191; 5-year event free survival EFS 0.84 +/- 0.03 v 0.38 +/- 0.04; 5-year overall survival OS 0.98 +/- 0.01 v 0.56 +/- 0.05, respectively; both P < .001). Moreover, patients with divergent outcome were robustly discriminated in both German and international cohorts and in prospectively analyzed samples (P <or= .001 for both EFS and OS for each). In subgroups with clinical low-, intermediate-, and high-risk of death from disease, the PAM predictor significantly separated patients with divergent outcome (low-risk 5-year OS: 1.0 v 0.75 +/- 0.10, P < .001; intermediate-risk: 1.0 v 0.82 +/- 0.08, P = .042; and high-risk: 0.81 +/- 0.08 v 0.43 +/- 0.05, P = .001). In multivariate Cox regression models based on both EFS and OS, PAM was a significant independent prognostic marker (EFS: hazard ratio HR, 3.375; 95% CI, 2.075 to 5.492; P < .001; OS: HR, 11.119, 95% CI, 2.487 to 49.701; P < .001). The highest potential clinical impact of the classifier was observed in patients currently considered as non-high-risk (n = 289; 5-year EFS: 0.87 +/- 0.02 v 0.44 +/- 0.07; 5-year OS: 1.0 v 0.80 +/- 0.06; both P < .001).
Gene expression-based classification using the 144-gene PAM predictor can contribute to improved treatment stratification of neuroblastoma patients.
Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to ...differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2′-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.
•Low TFAP2B transcript levels are associated with unfavorable prognostic markers and poor outcome in neuroblastoma.•TFAP2B may be silenced by promoter methylation in unfavorable neuroblastoma.•TFAP2B plays a role in neuronal differentiation of neuroblastoma cells.
Ecosystems are increasingly managed to provide multiple benefits to humans, which often degrades their ecological integrity. This strongly applies to aquatic ecosystems, in which engineering can ...enhance flood protection, drinking water supply, fisheries and recreation. Although these activities typically increase ecosystem functionality to humans, they often impair key aspects of biodiversity and natural functioning.
Classical restoration of such degrading freshwater ecosystems can lead to societal opposition, if returning to a former ecosystem state affects previously acquired ecosystem services. Innovative nature‐based solutions are therefore needed that enhance natural values in ecosystems, without affecting existing services.
We present a large‐scale project aiming to increase the ecological integrity of a human‐modified freshwater lake while maintaining its services to humans. The freshwater lake Markermeer in the Netherlands was formed by closing off an estuary for flood protection. The ecological integrity of this lake diminished over time, likely because a declining primary productivity impaired biodiversity at higher trophic levels. This decline is associated with a lack of gradual land–water transitions, strong resuspension of fine sediments, low nutrient availability and lack of dynamics typically to be expected in a natural temperate freshwater lake. Restoring the lake to its former marine state would conflict with current ecosystem services.
A nature‐based solution was initiated in 2016, consisting of constructing a five‐island archipelago from the lake's own soft‐sediments called the ‘Marker Wadden’. The project aims to increase the lake's primary production by creating gradual land–water transitions, more heterogeneity in water depths and decreasing turbidity by creating shelter and deep sinks reducing fine‐sediment resuspension by the wind – thus introducing currently missing elements that are typical for natural lakes. We present the underlying ecological framework and first scientific results of this innovative ongoing project.
Within 4 years, the Marker Wadden project shows how forward‐looking sustainable development of lake ecosystems using a rewilding approach can enhance natural processes and attract birds and fish, without conflicting with existing ecosystem services. This inspires new directions for halting and reversing the degradation of other vital ecosystems worldwide.
Samenvatting
Economische ontwikkelingen gaan vaak ten koste van de ecologische integriteit van ecosystemen. Dit geldt zeker voor zoetwater‐ecosystemen die worden ingericht ten behoeve van de waterveiligheid, drinkwatervoorziening, commerciële bevissing of recreatie. Maar wat voor mensen mogelijk een verbetering van de functionaliteit van een rivier of meer betekent, gaat vaak ten koste van de biodiversiteit en ruimte voor natuurlijke processen.
Herstel van beschadigde zoetwater‐ecosystemen op klassieke wijze, namelijk het terugkeren naar de situatie van voor het menselijk ingrijpen, kan maatschappelijke weerstand oproepen als dit ten koste gaat van die eerder verworven ecosysteem diensten. Het vergt innovatieve oplossingen gericht op natuurlijke processen om de natuurwaarden te verhogen en tegelijkertijd de functionaliteit voor de mens ervan te behouden.
Deze nieuwe vorm van ecosysteemherstel is toegepast in een grootschalig project in het Nederlandse Markermeer. Het Markermeer is in 1975 kunstmatig ontstaan in het voormalig estuarium van De Zuiderzee door bedijking voor de waterveiligheid. De afgelopen decennia is de ecologische waarde van het Markermeer steeds verder achteruitgegaan, waarschijnlijk doordat de primaire productie afneemt, met consequenties voor het hele voedsel web. Deze afname van productiviteit wordt geassocieerd met een gebrek aan natuurlijke land‐water overgangen, slecht doorzicht door continue opwerveling van grote hoeveelheden fijn slib, lage beschikbaarheid van voedingsstoffen en een gebrek aan natuurlijke dynamiek die past bij een natuurlijk gevormd ondiep zoetwatermeer. Klassiek herstel van de ecologische integriteit van het Markermeer via terugkeer naar de voormalige Zuiderzee is inmiddels onmogelijk omdat dit in strijd is met de huidige ecosysteemfuncties.
In 2016 is daarom begonnen met een innovatieve vorm van ecosysteemherstel: de bouw van een nieuwe archipel van vijf eilanden, de Marker Wadden. De eilanden hebben als doel de primaire productie van het Markermeer te stimuleren door het toevoegen van karakteristieke elementen van natuurlijke meren die op dit moment in het meer ontbreken. Dit zijn met name geleidelijke land‐waterovergangen, variatie in waterdieptes en luwten tussen de eilanden waar het door de wind opwervelende slib kan bezinken. Hier presenteren we de ecologische achtergrond en eerste wetenschappelijke bevindingen van dit unieke en innovatieve project.
Binnen vier jaar laat het Marker Wadden‐project zien hoe natuurontwikkeling volgens een rewilding benadering een stimulans kan geven aan natuurlijke processen. Vogels en vissen blijken het nieuwe gebied direct in gebruik te nemen, terwijl de ecosysteemfuncties voor de mens behouden zijn gebleven. Dit project kan dienen als voorbeeld van een nieuwe vorm van ecosysteemherstel, wat hard nodig is om de huidige achteruitgang van belangrijke ecosystemen wereldwijd ten goede te keren.
The Marker Wadden project shows how forward‐looking sustainable development of lake ecosystems using a rewilding approach can enhance natural processes and attract birds and fish, without conflicting with existing ecosystem services. This inspires new directions for halting and reversing the degradation of other vital ecosystems worldwide.
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