Accurate prediction of neuroblastoma outcome based on miRNA expression profiles

Abstract

For neuroblastoma, the most common extracranial tumour of childhood, identification of new biomarkers and potential therapeutic targets is mandatory to improve risk stratification and survival rates. MicroRNAs are deregulated in most cancers, including neuroblastoma. In this study, we analysed 430 miRNAs in 69 neuroblastomas by stem-loop RT-qPCR. Prediction of event-free survival (EFS) with support vector machines (SVM) and actual survival times with Cox regression-based models (CASPAR) were highly accurate and were independently validated. SVM-accuracy for prediction of EFS was 88.7% (95% CI: 88.5-88.8%). For CASPAR-based predictions, 5y-EFS probability was 0.19% (95% CI: 0-38%) in the CASPAR-predicted short survival group compared with 0.78% (95%CI: 64-93%) in the CASPAR-predicted long survival group. Both classifiers were validated on an independent test set yielding accuracies of 94.74% (SVM) and 5y-EFS probabilities as 0.25 (95% CI: 0.0-0.55) for short versus 1 6 0.0 for long survival (CASPAR), respectively. Amplification of the MYCN oncogene was highly correlated with deregulation of miRNA expression. In addition, 37 miRNAs correlated with TrkA expression, a marker of excellent outcome, and 6 miRNAs further analysed in vitro were regulated upon TrkA transfection, suggesting a functional relationship. Expression of the most significant TrkA-correlated miRNA, miR-542-5p, also discriminated between local and metastatic disease and was inversely correlated with MYCN amplification and event-free survival. We conclude that neuroblastoma patient outcome prediction using miRNA expression is feasible and effective. Studies testing miRNA-based predictors in comparison to and in combination with mRNA and aCGH information should be initiated. Specific miRNAs (e.g., miR-542-5p) might be important in neuroblastoma tumour biology, and qualify as potential therapeutic targets.Relapse or progression of advanced stage neuroblastoma (NB), the most common extracranial solid tumour of childhood, represents a major treatment challenge in paediatric oncology. Almost one third of all children with advanced stage NB is incurable, and NB accounts for approximately 15% of all childhood cancer deaths. Neuroblastoma is clinically heterogeneous. Tumours of patients with favourable disease may undergo spontaneous regression or differentiation even without therapy. In contrast, patients with unfavourable NB die despite intensive therapy. Favourable tumours are most often localised, stage 1 or 2 tumours, and unfavourable are most often stage 4, metastasised tumours. Stage 4 tumours are metastasised tumours in patients under 1 year of age, which often undergo spontaneous regression with no or minimal therapy. The tumour biology of unfavourable NB is characterised by MYCN amplification, low expression of the TrkA receptor tyrosine kinase and distinct chromosomal aberrations (reviewed in Refs. 1 and 2). Survival rates of patients with unfavourable NB can only be improved by a better understanding of the molecular pathogenesis of NB. This may...

N-myc and Noncoding RNAs in Neuroblastoma

“…Other studies have confirmed direct binding of N-myc to the mir-17-92 promoter (50, 55), as well as a positive correlation between expression of MYCN and members of the mir-17-92 cluster in primary tumors and/or neuroblastoma cell lines (49,51,(55)(56)(57)(58)(59)(60)(61). As miRNAs simultaneously target a variety of different mRNAs, it became clear that activation of the mir-17-92 cluster enables N-myc to turn multiple cellular processes toward malignant transformation.…”

“…Expression of mir-542-5p was nonrandomly distributed among tumor genetic subtypes, with lowest expression in MYCN-amplified tumors (77% completely lacking expression) and highest expression in stage 1,2,3 and 4S tumors (24% lacking mir-542-5p). Patients with tumors lacking mir-542-5p expression had the poorest prognosis independently of the MYCN status in the tumors (60,75). Bray and colleagues further showed that mir-542-5p overexpression in MYCN-amplified and nonamplified neuroblastoma cells reduced invasiveness in vitro, and restricted tumor growth and metastasis in vivo when cells were orthotopically injected into CB-17/SCID mice.…”

“…Several studies have shown an inverse correlation between mir-542-5p and MYCN amplification in primary tumors (49,51,60,75). In a large-scale profiling study of 430 miRNAs in 69 primary tumors, Schulte and colleagues (60) found increased expression of 4 miRNAs in Model for an N-myc-mir-9-E-cadherin pathway involved in metastasis formation. N-myc activates mir-9, which suppresses the expression of E-cadherin and promotes cell motility and invasiveness.…”

“…Although the expression of several miRNAs have been documented to correlate with MYCN expression, there is now growing evidence that N-myc predominantly acts repressive on the overall miRNA composition in MYCNamplified neuroblastoma cells (47,49,(59)(60)(61)71) and upon N-myc induction in nonamplified neuroblastoma cells (55). Lin and colleagues (71) used real-time RT-PCR to profile the expression of 162 miRNAs in 66 primary neuroblastoma tumors (including 13 with MYCN-amplification) and found a nearly global downregulation of miRNAs in high-risk tumors, especially in those with MYCN amplification.…”

MicroRNAs-10a and -10b Contribute to Retinoic Acid-induced Differentiation of Neuroblastoma Cells and Target the Alternative Splicing Regulatory Factor SFRS1 (SF2/ASF)

“…All these results prompted the introduction of RA and its derivatives into therapeutic protocols for neuroblastoma patients, with some success especially in the treatment of minimal residual disease (24 -26). Although several evidences support a role for miRNAs in neuroblastoma pathogenesis (27)(28)(29)(30) and the usefulness of miRNA profiles for neuroblastoma diagnostics, classification, and prognosis has been recently reported (31)(32)(33), little is known about the involvement of miRNAs in RA-induced differentiation of neuroblastoma cells.…”