Jessica Theissen

Telomerase activation by genomic rearrangements in high-risk neuroblastoma

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

Prognostic Impact of Gene Expression–Based Classification for Neuroblastoma

PURPOSE To evaluate the impact of a predefined gene expression-based classifier for clinical risk estimation and cytotoxic treatment decision making in neuroblastoma patients. PATIENTS AND METHODS 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. RESULTS 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). CONCLUSION Gene expression-based classification using the 144-gene PAM predictor can contribute to improved treatment stratification of neuroblastoma patients.

Mutational dynamics between primary and relapse neuroblastomas

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.

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival

Several microRNA (miRNA) loci are found within genomic regions frequently deleted in primary neuroblastoma, including miR-885-5p at 3p25.3. In this study, we demonstrate that miR-885-5p is downregulated on loss of 3p25.3 region in neuroblastoma. Experimentally enforced miR-885-5p expression in neuroblastoma cell lines inhibits proliferation triggering cell cycle arrest, senescence and/or apoptosis. miR-885-5p leads to the accumulation of p53 protein and activates the p53 pathway, resulting in upregulation of p53 targets. Enforced miR-885-5p expression consistently leads to downregulation of cyclin-dependent kinase (CDK2) and mini-chromosome maintenance protein (MCM5). Both genes are targeted by miR-885-5p via predicted binding sites within the 3′-untranslated regions (UTRs) of CDK2 and MCM5. Transcript profiling after miR-885-5p introduction in neuroblastoma cells reveals alterations in expression of multiple genes, including several p53 target genes and a number of factors involved in p53 pathway activity. Taken together, these data provide evidence that miR-885-5p has a tumor suppressive role in neuroblastoma interfering with cell cycle progression and cell survival.

Comparison of RNA-seq and microarray-based models for clinical endpoint prediction

BackgroundGene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model.ResultsWe generate gene expression profiles from 498 primary neuroblastomas using both RNA-seq and 44 k microarrays. Characterization of the neuroblastoma transcriptome by RNA-seq reveals that more than 48,000 genes and 200,000 transcripts are being expressed in this malignancy. We also find that RNA-seq provides much more detailed information on specific transcript expression patterns in clinico-genetic neuroblastoma subgroups than microarrays. To systematically compare the power of RNA-seq and microarray-based models in predicting clinical endpoints, we divide the cohort randomly into training and validation sets and develop 360 predictive models on six clinical endpoints of varying predictability. Evaluation of factors potentially affecting model performances reveals that prediction accuracies are most strongly influenced by the nature of the clinical endpoint, whereas technological platforms (RNA-seq vs. microarrays), RNA-seq data analysis pipelines, and feature levels (gene vs. transcript vs. exon-junction level) do not significantly affect performances of the models.ConclusionsWe demonstrate that RNA-seq outperforms microarrays in determining the transcriptomic characteristics of cancer, while RNA-seq and microarray-based models perform similarly in clinical endpoint prediction. Our findings may be valuable to guide future studies on the development of gene expression-based predictive models and their implementation in clinical practice.

Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma

Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression.

High ALK Receptor Tyrosine Kinase Expression Supersedes ALK Mutation as a Determining Factor of an Unfavorable Phenotype in Primary Neuroblastoma

Purpose: 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. Experimental Design: 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. Results: 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. Conclusions: 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. Clin Cancer Res; 17(15); 5082–92. ©2011 AACR.

Heterogeneity of the MYCN Oncogene in Neuroblastoma

Purpose:MYCN amplification is an important therapy-stratifying marker in neuroblastoma. Fluorescence in situ hybridization with signal detection on the single-cell level allows a critical judgement of MYCN intratumoral heterogeneity. Experimental Design: The MYCN status was investigated by fluorescence in situ hybridization at diagnosis and relapse. Heterogeneity was defined as the simultaneous presence of amplified cells (≥5 cells per slide) and nonamplified cells within one tumor or sequential change of the amplification status during the course of the disease. Likewise, heterogeneity can be detected between primary tumor and metastasis. Results: From 1,341 patients analyzed, 1,071 showed no amplification, 250 showed homogeneous amplification, and 20 patients showed MYCN heterogeneity. Of the patients with heterogeneity, 12 of 20 had clusters of MYCN amplifications, 3 of 20 had amplified single cells, 3 of 20 showed MYCN amplifications in the bone marrow but not in the primary tumor, and 2 of 20 acquired MYCN amplification during the course of the disease. All stage 4 patients were treated according to high-risk protocols; 7 of 8 later progressed. Four patients with localized disease were treated according to high-risk protocol because of MYCN-amplified clusters; 1 of 4 later progressed. One patient treated with mild chemotherapy experienced progression. Seven patients with localized/4S disease underwent no chemotherapy: 4 of 5 patients with MYCN heterogeneity at diagnosis remained disease-free, and 1 of 5 experienced local progression. Two patients had normal MYCN status at diagnosis but acquired MYCN amplification during the course of the disease. Conclusion:MYCN heterogeneity is rare. Our results suggest that small amounts of MYCN-amplified cells are not correlated to adverse outcomes. More patients with heterogeneity are warranted to clarify the role of MYCN heterogeneity for risk classification.

Integrated genomic profiling identifies two distinct molecular subtypes with divergent outcome in neuroblastoma with loss of chromosome 11q.

Imbalances in chromosome 11q occur in approximately 30% of primary neuroblastoma and are associated with poor outcome. It has been suggested that 11q loss constitutes a distinct clinico-genetic neuroblastoma subgroup by affecting expression levels of corresponding genes. This study analysed the relationship of 11q loss, clinical phenotype and global transcriptomic profiles in four clinico-genetic subgroups (11q alteration/favourable outcome, n=7; 11q alteration/unfavourable outcome, n=14; no 11q alteration/favourable outcome, n=81; no 11q alteration/unfavourable outcome, n=8; tumours with MYCN amplification and/or 1p loss were excluded). Unsupervised and supervised comparisons of gene expression profiles consistently showed significantly different mRNA patterns between favourable and unfavourable neuroblastomas, both in the subgroups with and without 11q loss. In contrast, favourable tumours with and without 11q loss showed highly similar transcriptomic profiles. Disproportionate downregulation of 11q genes was observed only in unfavourable tumours with 11q loss. The diverging molecular profiles were neither caused by considerable differences in the size of the deleted regions nor by differential methylation patterns of 11q genes. Together, this study shows that neuroblastoma with 11q loss comprises two biological subgroups that differ both in their clinical phenotype and gene expression patterns, indicating that 11q loss is not a primary determinant of neuroblastoma tumour behaviour.

Molecular characterization and classification of neuroblastoma.

For many decades, neuroblastoma has remained a challenging disease for both clinicians and researchers. Now, techniques that efficiently specify both comprehensive genetic and gene-expression alterations of neuroblastoma tumors have provided molecular markers that indicate tumor behavior and patient outcome with very high accuracy. Once the anticipated value of these markers has been confirmed in ongoing studies, patients may profit from more accurate risk assessment by integrating these markers into clinical routine. Moreover, disclosing further tumor-initiating events, such as the recently revealed oncogenic mutations of ALK, will further promote the elucidation of the genetic etiology of the disease. Together with recent information on altered signaling pathways in aggressively growing tumors, this knowledge will help to establish therapeutic strategies specifically targeting molecular key factors of neuroblastoma tumor progression.