Osteosarcoma-Specific Genes as a Diagnostic Tool and Clinical Predictor of Tumor Progression

SIMPLE SUMMARY: The standard method for the diagnosis and monitoring of osteosarcoma is biopsy and tumor imaging, which causes discomfort to patients and is difficult to repeat. A blood sample can be used as a non-invasive method for monitoring tumor material. Vimentin and ezrin show clinical significance in samples obtained from OS patients but need circulating tumor cell purification, since they are expressed in leukocytes. Due to the low-temperature storage of the samples, it proved impossible to perform purification to remove the contamination. We propose that novel or OS-specific biomarkers using differential gene expression from the Gene Expression Omnibus (GEO) database is a promising approach for developing diagnostic and tumor progression strategies. Seven genes from the database showed significant expression in OS cell lines/primary cells compared to a normal blood donor, together with ezrin and VIM. The expression of the five candidate genes together with ezrin and vimentin were quantified by qRT-PCR and analyzed using a mathematical model with high efficiency to discriminate between OS patients and normal samples, resulting in the selection of three candidate genes: COL5A2 (one of the five from the database) as well as ezrin and VIM. Our study demonstrates that these genes in retrospective samples could serve as tools of OS detection and predictors of disease progression. ABSTRACT: A liquid biopsy is currently an interesting tool for measuring tumor material with the advantage of being non-invasive. The overexpression of vimentin and ezrin genes was associated with epithelial-mesenchymal transition (EMT), a key process in metastasis and progression in osteosarcoma (OS). In this study, we identified other OS-specific genes by calculating differential gene expression using the Gene Expression Omnibus (GEO) database, confirmed by using quantitative reverse transcription-PCR (qRT-PCR) to detect OS-specific genes, including VIM and ezrin in the buffy coat, which were obtained from the whole blood of OS patients and healthy donors. Furthermore, the diagnostic model for OS detection was generated by utilizing binary logistic regression with a multivariable fractional polynomial (MFP) algorithm. The model incorporating VIM, ezrin, and COL5A2 genes exhibited outstanding discriminative ability, as determined by the receiver operating characteristic curve (AUC = 0.9805, 95% CI 0.9603, 1.000). At the probability cut-off value of 0.3366, the sensitivity and the specificity of the model for detecting OS were 98.63% (95% CI 90.5, 99.7) and 94.94% (95% CI 87.5, 98.6), respectively. Bioinformatic analysis and qRT-PCR, in our study, identified three candidate genes that are potential diagnostic and prognostic genes for OS.