Molecular Subtyping and Survival Analysis of Osteosarcoma Reveals Prognostic Biomarkers and Key Canonical Pathways

Abstract

Osteosarcoma (OS) is a common bone malignancy in children and adolescents. Although histological subtyping followed by improved OS treatment regimens have helped achieve favorable outcomes, a lack of understanding of the molecular subtypes remains a challenge to characterize its genetic heterogeneity and subsequently to identify diagnostic and prognostic biomarkers for developing effective treatments. In the present study, global analysis of DNA methylation, and mRNA and miRNA gene expression in OS patient samples were correlated with their clinical characteristics. The mucin family of genes, MUC6, MUC12, and MUC4, were found to be highly mutated in the OS patients. Results revealed the enrichment of molecular pathways including Wnt signaling, Calcium signaling, and PI3K-Akt signaling in the OS tumors. Survival analyses showed that the expression levels of several genes such as RAMP1, CRIP1, CORT, CHST13, and DDX60L, miRNAs and lncRNAs were associated with survival of OS patients. Molecular subtyping using Cluster-Of-Clusters Analysis (COCA) for mRNA, lncRNA, and miRNA expression; DNA methylation; and mutation data from the TARGET dataset revealed two distinct molecular subtypes, each with a distinctive gene expression profile. Between the two subtypes, three upregulated genes, POP4, HEY1, CERKL, and seven downregulated genes, CEACAM1, ABLIM1, LTBP2, ISLR, LRRC32, PTPRF, and GPX3, associated with OS metastasis were found to be differentially regulated. Thus, the molecular subtyping results provide a strong basis for classification of OS patients that could be used to develop better prognostic treatment strategies.

Keywords: COCA; Cluster-Of-Clusters Analysis; DNA methylation; TARGET database; classification of osteosarcoma; mRNA and miRNA expression; multiomics data analysis.

Figure 1

(A) Oncoplot showing the top 5 altered genes with more than 10% samples containing mutations in the TCGA enriched pathways. The fraction and the number of samples with variations in the corresponding genes are shown on the right as percentages and the bar plots (B) Enriched oncogenic pathways showing the fraction of genes and samples affected.

Figure 2

Kaplan-Meier plot for high vs low expression group in TARGET OS data with p-value from log-rank test and Cox regression model for genes, METTL20, MYH10, and PDE1B with HR < 1 (A), and genes, RAMP1, TAC4, and MT1 with HR > 1 (B).

Figure 3

(A) Correlation plot showing significantly correlated canonical pathway genes (p < 0.05). Negative correlated genes are shown in red, and positive in blue. (B) List of 34 genes found significantly associated with survival (p < 0.05), their associated canonical pathways, and corresponding HR values.

Figure 4

Average silhouette width and NMF classification with rank using 190 miRNAs, 1107 mutations, 2500 protein-coding regions, 500 lncRNA, and 5000 CpG methylation probe data for 85 OS-TARGET patients which was further used as input for COCA for obtaining the final two molecular subtypes.

Figure 5

(A) Volcano plot showing the differentially expressed genes between the two molecular subtypes. The downregulated (green) and upregulated (red) genes obtained at abs(log₂Fold Change) > 1.5 and adjusted p-value < 0.05 are highlighted. (B) Heatmap showing top 100 most variable differentially expressed genes obtained at abs(log₂Fold Change) > 1 and adjusted p-value < 0.01 cutoff found between the two molecular clusters.