Abstract
BackgroundA microRNA (miRNA) collection on the imprinted 14q32 MEG3 region has been associated with outcome in osteosarcoma. We assessed the clinical utility of this miRNA set and their association with methylation status.MethodsWe integrated coding and non-coding RNA data from three independent annotated clinical osteosarcoma cohorts (n = 65, n = 27, and n = 25) and miRNA and methylation data from one in vitro (19 cell lines) and one clinical (NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) osteosarcoma dataset, n = 80) dataset. We used time-dependent receiver operating characteristic (tdROC) analysis to evaluate the clinical value of candidate miRNA profiles and machine learning approaches to compare the coding and non-coding transcriptional programs of high- and low-risk osteosarcoma tumors and high- versus low-aggressiveness cell lines. In the cell line and TARGET datasets, we also studied the methylation patterns of the MEG3 imprinting control region on 14q32 and their association with miRNA expression and tumor aggressiveness.ResultsIn the tdROC analysis, miRNA sets on 14q32 showed strong discriminatory power for recurrence and survival in the three clinical datasets. High- or low-risk tumor classification was robust to using different microRNA sets or classification methods. Machine learning approaches showed that genome-wide miRNA profiles and miRNA regulatory networks were quite different between the two outcome groups and mRNA profiles categorized the samples in a manner concordant with the miRNAs, suggesting potential molecular subtypes. Further, miRNA expression patterns were reproducible in comparing high-aggressiveness versus low-aggressiveness cell lines. Methylation patterns in the MEG3 differentially methylated region (DMR) also distinguished high-aggressiveness from low-aggressiveness cell lines and were associated with expression of several 14q32 miRNAs in both the cell lines and the large TARGET clinical dataset. Within the limits of available CpG array coverage, we observed a potential methylation-sensitive regulation of the non-coding RNA cluster by CTCF, a known enhancer-blocking factor.ConclusionsLoss of imprinting/methylation changes in the 14q32 non-coding region defines reproducible previously unrecognized osteosarcoma subtypes with distinct transcriptional programs and biologic and clinical behavior. Future studies will define the precise relationship between 14q32 imprinting, non-coding RNA expression, genomic enhancer binding, and tumor aggressiveness, with possible therapeutic implications for both early- and advanced-stage patients.
Highlights
A microRNA collection on the imprinted 14q32 MEG3 region has been associated with outcome in osteosarcoma
For most of the MEG3 CpG sites targeted by methylation probes, we found remarkable similarity between the moderate or moderately strong methylation/miRNA correlations observed in the cell lines (Table 2) and the correlations seen in the TARGET data (Fig. 10)
Using the largest osteosarcoma cell line dataset with genome-wide molecular information, we found that the expression patterns of the 14q32 miRNAs as a function of cell line aggressiveness were largely concordant with the findings in the clinical datasets
Summary
A microRNA (miRNA) collection on the imprinted 14q32 MEG3 region has been associated with outcome in osteosarcoma. We recently described miRNA expression models for recurrence and overall survival from formalin-fixedparaffin-embedded (FFPE) biopsy specimens that confer strong prognostic discrimination independent of chemotherapy response [6]. The majority of these prognostic miRNAs are located on the 14q32 locus, one of the few genomic regions that are imprinted in normal cells, which is thought to be critical in tissue development via a tightly controlled, allele-specific DNA methylation effect on gene expression. Our findings suggest that the non-coding 14q32 cluster contains a large number of useful clinically relevant biomarkers and is a locus of substantial genomic and epigenetic alterations that give rise to novel subtypes of osteosarcoma with distinct clinical, molecular, and biological context and therapeutic implications
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