mRNA expression profiles of primary high-grade central osteosarcoma are preserved in cell lines and xenografts

Abstract

Background: Conventional high-grade osteosarcoma is a primary malignant bone tumor, which is most prevalent in adolescence. Survival rates of osteosarcoma patients have not improved significantly in the last 25 years. Aiming to increase this survival rate, a variety of model systems are used to study osteosarcomagenesis and to test new therapeutic agents. Such model systems are typically generated from an osteosarcoma primary tumor, but undergo many changes due to culturing or interactions with a different host species, which may result in differences in gene expression between primary tumor cells, and tumor cells from the model system. We aimed to investigate whether gene expression profiles of osteosarcoma cell lines and xenografts are still comparable to those of the primary tumor.

Methods: We performed genome-wide mRNA expression profiling on osteosarcoma biopsies (n = 76), cell lines (n = 13), and xenografts (n = 18). Osteosarcoma can be subdivided into several histological subtypes, of which osteoblastic, chondroblastic, and fibroblastic osteosarcoma are the most frequent ones. Using nearest shrunken centroids classification, we generated an expression signature that can predict the histological subtype of osteosarcoma biopsies.

Results: The expression signature, which consisted of 24 probes encoding for 22 genes, predicted the histological subtype of osteosarcoma biopsies with a misclassification error of 15%. Histological subtypes of the two osteosarcoma model systems, i.e. osteosarcoma cell lines and xenografts, were predicted with similar misclassification error rates (15% and 11%, respectively).

Conclusions: Based on the preservation of mRNA expression profiles that are characteristic for the histological subtype we propose that these model systems are representative for the primary tumor from which they are derived.

Figure 1

Subtype-specific genes. Venn diagram representing numbers of fibroblastic- (green), chondroblastic- (red), and osteoblastic (blue)-specific differentially expressed genes obtained with factorial LIMMA analysis, considering chondroblastic versus osteoblastic (chondro vs osteo), fibroblastic versus osteoblastic (fibro vs osteo), and chondroblastic versus fibroblastic (chondro vs fibro) analyses. Subtype-specific genes are genes that are either both upregulated or both downregulated in the subtype of interest in the different comparisons.

Figure 2

Nearest shrunken centroids classification. A Illustration of training the pamr prediction profile on osteosarcoma biopsies. At thresholds of 4.9-5.1, the misclassification error rate was minimal. B True versus predicted values from the nearest shrunken centroid fit. C Probabilities of each biopsy to belong to any of the three histological subtypes. Samples are separated (dotted lines) based on their true subtypes. Cross-validated probabilities for each histological subtype are shown on the y-axis, so that for every sample three open dots are present (blue, red, and green dots for osteo-, chondro-, and fibroblastic osteosarcoma, respectively). A sample is classified into a specific subtype if the probability to belong to that specific subtype is higher than the probabilities to belong to the other subtypes. D The FDR plotted against different thresholds of the prediction profile. At a threshold of 5.0, 24 genes are included in the prediction profile. These 24 genes have a FDR < 5%.

Figure 3

The prediction profile applied on cell lines and xenografts. Probabilities of A cell lines and B xenografts to belong to any of the three histological subtypes. For an explanation of what is represented by these graphs, see Figure 2C.