BMC Cancer 2009, 9:11 http://www.biomedcentral.com/1471-2407/9/11 sibly due to the heterogeneous nature of the samples as well as the relatively small number of samples in this study (Figure 2). Assessment of correlation with lymph node status, stage and outcome We grouped patients with pathologic stages I and II dis- ease into an early-stage disease and grouped patients with pathologic stages III and IV disease into a late-stage dis- ease category. Through statistical regression analysis, we identified genes whose expression differed in tumor ver- sus normal mucosa and those whose expression was most different between the staging subgroups (Table 3A). The same analysis was performed to compare patients without cervical nodal metastasis (N0) to those with nodal disease (N1–N3) (Table 3B). We analyzed data from patients (n = 20) for which tumor and matched normal mucosae were available and not the larger subgroup of patients (n = 37) in order to obtain a more meaningful comparison of gene expression changes between tumor and normal mucosa. We selected three genes, GLUT3, HSAL2 and PACE4 for further analysis and validation in a larger cohort of 49 patients. We selected genes with known important roles in cellular functions and carcinogenesis and for which anti- bodies were available. We employed a two-step quantita- tive RT-PCR to validate expression changes identified by gene array analysis for the three selected genes in all 49 cases. We defined the cut-off value for over-expression as two-fold or greater relative to matched normal controls. Using these criteria, 30.6%, 24.5% and 26.5% of patients expressed high levels of GLUT3, HSAL2 and PACE4, respectively. We assessed the prognostic significance of expression of the selected genes and various clinicopatho- logical parameters. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P < 0.0001), tumor size (P = 0.024), pathological stage (P = 0.009) and recurrence (P = 0.038). HSAL2 was posi- tively associated with depth of invasion (P = 0.015) and advanced T stage (P = 0.047). PACE4 expression failed to show correlation with clinicopathological parameters. Table 4 depicts the univariate analysis of GLUT3, HSAL2 and PACE4 expression and various clinicopathologic parameters. In survival studies, only GLUT3 showed a prognostic value with disease-free survival (P = 0.049), relapse-free survival (P = 0.002) and overall survival (P = 0.003) (Figure 3). Multivariate analysis with Cox's pro- portional hazards revealed that all parameters remained independent prognosticators in this group of patients. B Page 8 of 13 (page number not for citation purposes) aaaa A FHiigeurarrech2ical clustering of tumor (T), N = 31 Hierarchical clustering of tumor (T), N = 31. A; and normal (N), N = 26. B cluster sets based on the complete panel of 12,000 genes. Horizontal distance represents the correlation of the samples to one another. The numbers at each node repre- sent frequency of the shown pattern using 1,000 iteration bootstrap resampling.