Microarray analysis identifies distinct gene expression profiles associated with histological subtype in human osteosarcoma

Abstract

Osteosarcoma is the most common primary malignant bone tumour. Currently osteosarcoma classification is based on histological appearance. It was the aim of this study to use a more systematic approach to osteosarcoma classification based on gene expression analysis and to identify subtype specific differentially expressed genes. We analysed the global gene expression profiles of ten osteosarcoma samples using Affymetrix U133A arrays (five osteoblastic and five non-osteoblastic osteosarcoma patients). Differential gene expression analysis yielded 75 genes up-regulated and 97 genes down-regulated in osteoblastic versus non-osteoblastic osteosarcoma samples, respectively. These included genes involved in cell growth, chemotherapy resistance, angiogenesis, steroid- and neuropeptide hormone receptor activity, acute-phase response and serotonin receptor activity and members of the Wnt/ß-catenin pathway and many others. Furthermore, we validated the highly differential expression of six genes including angiopoietin 1, IGFBP3, ferredoxin 1, BMP, decorin, and fibulin 1 in osteoblastic osteosarcoma relative to non-osteoblastic osteosarcoma. Our results show the utility of gene expression analysis to study osteosarcoma subtypes, and we identified several genes that may play a role as potential therapeutic targets in the future.

Fig. 1

Heat map and supervised hierarchical clustering of genes that distinguish osteoblastic from non-osteoblastic osteosarcoma patients. Each row represents a gene, and patient samples are depicted in columns. Red indicates genes that are expressed at higher levels. Green indicates genes that are expressed at lower levels compared with mean expression

Fig. 2

Functional and pathway classification of the differentially expressed genes. Columns represent the gene expression levels of osteoblastic osteosarcoma patients (group A) and non-osteoblastic osteosarcoma patients (group B). Gene expression profile through pathway analysis demonstrates that genes involved in nucleobase- and polyamine metabolism and aerobic respiration were up-regulated in osteoblastic osteosarcoma patients (group A). Genes involved in steroid- and neuropeptide hormone receptor activity, acute-phase response and serotonin receptor activity were upregulated in non-osteoblastic osteosarcoma patients (group B). Each row represents a gene, and patient samples are depicted in columns. Red indicates genes that are expressed at higher levels. Green indicates genes that are expressed at lower levels compared with mean expression

Fig. 3

Real-time (RT)-PCR for angiopoietin (a), IGFBP-3 (b), ferredoxin 1 (c), BMP (d), decorin (e), and fibulin 1 (f) was performed on total RNA extracted from tumour biopsies deriving from the malignant tissue of five patients with osteoblastic osteosarcoma and from five patients with non-osteoblastic osteosarcoma. Differences in gene expression levels between the two groups were analysed by Student’s t test. Primers and cycling conditions for each of the amplified genes are described in Materials and Methods