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. 2009 Nov 16;4(11):e7850.
doi: 10.1371/journal.pone.0007850.

Widespread dysregulation of MiRNAs by MYCN amplification and chromosomal imbalances in neuroblastoma: association of miRNA expression with survival

Isabella Bray  1 , Kenneth Bryan, Suzanne Prenter, Patrick G Buckley, Niamh H Foley, Derek M Murphy, Leah Alcock, Pieter Mestdagh, Jo Vandesompele, Frank Speleman, Wendy B London, Patrick W McGrady, Desmond G Higgins, Anne O'Meara, Maureen O'Sullivan, Raymond L Stallings
Affiliations

Widespread dysregulation of MiRNAs by MYCN amplification and chromosomal imbalances in neuroblastoma: association of miRNA expression with survival

Isabella Bray et al. PLoS One. .

Abstract

MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Two-way hierarchical cluster analysis on the training set of tumors (n = 96) using 37 miRNAs that were significantly differentially expressed between MNA and non-MNA tumors.
Tumor cluster A was significantly enriched for MNA tumors (69%). The Kaplan-Meier graphs at the bottom of the figure show that EFS and OS was significantly reduced in the MNA tumor cluster (Cluster A) relative to the non-MNA cluster (Cluster B).
Figure 2
Figure 2. Two-way hierarchical cluster analysis on the validation set of tumors (n = 48) using 37 miRNAs that were significantly differentially expressed between MNA and non-MNA tumors.
Tumor cluster A was significantly enriched for stage 4 tumors with MNA tumors. The Kaplan-Meier graphs at the bottom of the figure show that EFS and OS was significantly reduced in the MNA tumor cluster (Cluster A) relative to the non-MNA cluster (Cluster B).
Figure 3
Figure 3. Two-way hierarchical cluster analysis on the training set of tumors (n = 96) using the top 15 miRNAs that were significantly associated with OS, as determined by the Random Forest classifier.
As illustrated by the Kaplan-Meier graphs at the bottom of the figure, the patients from the two major cluster (A and B) differed dramatically in both EFS and OS. Cluster A is highly enriched for stage 4 tumors with MNA or loss of 11q and for patients with EFS and OS events. Notably, subclusters (B1 and B2) within the major cluster B showed a minor, but statistically significant difference in both EFS and OS.
Figure 4
Figure 4. Two-way hierarchical cluster analysis on the validation set of tumors (n = 48) using the top 15 miRNAs that were significantly associated with OS, as determined by the Random Forest classifier.
Similar to the training set of tumors, cluster A is highly enriched for stage 4 tumors with MNA or loss of 11q and for patients with EFS and OS events. As illustrated by the Kaplan-Meier graphs at the bottom of the figure, the patients from the two major clusters (A and B) differed dramatically in both EFS and OS. Notably, subclusters (B1 and B2) within the major cluster B showed a minor, but statistically significant difference in both EFS and OS.
Figure 5
Figure 5. Representative box-plots illustrating correlation between miRNA expression and DNA copy number status.
MiRNA locus, map location and p-value are displayed at the top of each box-plot while number of tumors having deletion, gain or normal DNA copy number status are displayed at the bottom. A through C are examples of miRNA expressional changes for regions that were both lost and gained in tumors. D through G are miRNA expressional changes for regions that showed only loss in the tumors, while H is an example of a miRNA mapping to a region that only showed gain in the entire tumor cohort. In the examples A through I miRNA expressional change is concordant with DNA copy number status, while box-plot I shows an example (miR-29c) of discordance between expression and copy number (i.e. tumors showing DNA copy number gain had significantly lower expression).
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References

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