Examination of oral cancer biomarkers by tissue microarray analysis

Objective: To validate the DNA microarray results on a subset of genes that could potentially serve as biomarkers of oral squamous cell carcinoma (OSCC) by examining their expression with an alternate quantitative method and by assessing their protein levels. Design: Based on DNA microarray data from our laboratory and data reported in the literature, we identified 6 potential biomarkers of OSCC to investigate further. We used quantitative real-time polymerase chain reaction to examine expression changes of CDH11, MMP3, SPARC, POSTN, TNC, and TGM3 in OSCC and histologically normal control tissues. We further examined validated markers at the protein level by immunohistochemical analysis of OSCC tissue microarray sections. Results: Quantitative real-time polymerase chain reaction analysis revealed upregulation of CDH11, SPARC, POSTN, and TNC gene expression and decreased TGM3 expression in OSCC tissue compared with control tissue; MMP3 was not found to be differentially expressed. In tissue microarray immunohistochemical analyses, SPARC (secreted protein, acidic, rich in cysteine), periostin, and tenascin C exhibited increased protein expression in tumor tissue compared with control tissue, and their expression was primarily localized within tumor-associated stroma rather than tumor epithelium. Conversely, transglutaminase 3 protein expression was found only within keratinocytes in control tissue and was significantly downregulated in cancer cells. Conclusions: Of 6 potential gene markers of OSCC, initially identified by DNA microarray analyses, differential expression of CDH11, SPARC, POSTN, TNC, and TGM3 were validated by quantitative real-time polymerase chain reaction. Differential expression and localization of proteins encoded by SPARC, POSTN, TNC, and TGM3 were clearly shown by tissue microarray immunohistochemical analysis.