Gene expression profiling to predict recurrence of advanced squamous cell carcinoma of the tongue: discovery and external validation

// Tomohiro Enokida 1, 2 , Satoshi Fujii 3 , Mari Takahashi 4 , Youichi Higuchi 3 , Shogo Nomura 5 , Tetsuro Wakasugi 1 , Tomoko Yamazaki 1 , Ryuichi Hayashi 6 , Atsushi Ohtsu 2, 7 and Makoto Tahara 1 1 Department of Head and Neck Medical Oncology, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan 2 Advanced Clinical Research of Cancer, Juntendo University Graduate School of Medicine, Bunkyo-Ku, Tokyo 113-8421, Japan 3 Division of Pathology, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Kashiwa, Chiba 277-8577, Japan 4 Department of Digestive Endoscopy, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan 5 Biostatistics Division, Center for Research Administration and Support, National Cancer Center, Kashiwa, Chiba 277-8577, Japan 6 Head and Neck Surgery Division, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan 7 National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan Correspondence to: Makoto Tahara, email: matahara@east.ncc.go.jp Satoshi Fujii, email: sfujii@east.ncc.go.jp Keywords: tongue squamous cell carcinoma, oral squamous cell carcinoma, gene expression profile, keratin 4, cytokeratin 4 Received: January 10, 2017     Accepted: May 23, 2017     Published: June 27, 2017 ABSTRACT Objectives: To establish a prognostic signature for locally advanced tongue squamous cell carcinoma (TSCC) patients treated with surgery. Results: In the discovery study, unsupervised hierarchical clustering analysis identified two clusters which differentiated the Kaplan-Meier curves of RFS [median RFS, 111 days vs. not reached; log-rank test, P = 0.023]. The 30 genes identified were combined into a dichotomous PI. In the validation cohort, classification according to the PI was associated with RFS [median RFS, 754 days vs. not reached; log-rank test, P = 0.026 in GSE31056] and DSS [median DSS, 540 days vs. not reached; log-rank test, P = 0.046 in GSE42743 and 443 days vs. not reached; P < 0.001 in GSE41613]. Among genes, positive immunohistochemical staining of cytokeratin 4 was associated with favorable prognostic values for RFS (hazard ratio (HR), 0.591, P = 0.045) and DSS (HR, 0.333, P = 0.004). Materials and methods: We conducted gene expression profiling of 26 clinicopathologically homogeneous advanced TSCC tissue samples using cDNA microarray as a discovery study. Candidate genes were screened using clustering analysis and univariate Cox regression analysis for relapse-free survival (RFS). These were combined into a prognostic index (PI), which was validated using three public microarray datasets of tongue and oral cancer (123 patients). Some genes identified in discovery were immunohistochemically examined for protein expression in another 127 TSCC patients. Conclusion: We identified robust molecular markers that showed significant associations with prognosis in TSCC patients. Gene expression profiling data were successfully converted to protein expression profiling data.