Yu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:119 http://www.jeccr.com/content/29/1/119 Page 5 of 9 45 25 101 10 67 26 x2 P 6.150 0.013 52.542 < 0.000 0.469 0.493 Variables Slug Cancer(120) Background(42) Snail cancer(120) Background(42) Twist cancer(120) Background(42) 75 17 19 32 53 16 Table 2 Expression and Snail, Slug, Twist and E-cadherin in bladder cancer and background tissue Positive Low expression (n) expression (n) and invasive BT (≥T2) (34 out of 44: 77.27%), LG (8 out of 41:19.51%), and HG (45 out of 79: 56.96%). The Twist immunoreactivity categorized into negative (< 2% of positive cells) vs. high expression was associated with several clinicopathological parameters: stage, grade, car- cinoma in situ (CIS), progression(Table 3). In the pT1 BT group, the high-risk pT1b (lamina propria invasion) showed a Twist overexpression almost similar to inva- sive BT, explaining that the prognostic of both types of tumor is quite the same(date not showed). Of the 120 BT specimens, we demonstrated no correlation between Slug and Snail expression and stage and grade, but we demonstrated a correlation between E-cadherin expres- sion and grade (p = 0.017): LMP (17 out of 18: 94.44%), LG (25 out of 41: 60.98%), and HG(64 out of 79: 81.01%) (Table 4). Only Twist was associated with clini- copathological parameters as progression(p = 0.035) (Table 4). It is interesting to note that Slug, Snail and E- cadherin had increased expression in node-positive tumors compared with node-negative tumors, these data reached significance(P = 0.012, P = 0.000, P = 0.040), respectively (Table 4). Twist was increased in node-posi- tive tumors (node positive,10/18; node negative,43/102 p = 0.291), although the value was not significantly different. Table 3 Correlation between E-cadherin expression and Snail, Slug, and Twist expression in 120 cases of bladder cancer E-cadherin expression(n) X2 P Slug expression(n) +(n = 75) -(n = 45) Twist expression(n) +(n = 53) -(n = 67) Snail expression(n) +(n = 19) -(n = 101) +(n = 89) 64 25 46 43 11 79 -(n = 31) 11 20 7 24 8 22 13.016 0.000 7.898 0.005 3.523 0.061 Correlation between proteins expression and BT recurrence During the follow-up period (median follow-up time 30 months (1-89), the total number of cases in which recurrence was observed is 36(30%) between 2 to 62 months after initial diagnosis. We failed to demonstrate any significant association between this event and the clinicopathological data tested or the Slug, Twist, Snail and E-cadherin expression. Even in the univariate and multivariate analyses. Correlation of proteins expression with survival of patients with BT-Univariate analysis Snail, Slug, Twist and E-cadherin is suggested to have a critical impact on progression and metastasis develop- ment as it positively influences the entrance of the tumor cells into the circulation (intravasation)[18-22]. To investigate the progression-free survival(PFS) or the overall survival (OS), we defined a time point of 36 months. During the time study, the 3-year PFS rates of patients who were positive (n = 53) and negative (n = 67) for Twist expression were 40% and 60%, posi- tive (n = 75) and negative (n = 45) for Slug expression were 61% and 39%, and positive (n = 19) and negative (n = 101) for Snail expression were 15% and 85%, respectively (Table 4). In univariate analysis, positive expression of Twist, Snail and loss of E-cadherin expres- sion, the stage, the grade, and CIS were significant pre- dictors of short PFS. But positive expression of Slug was not significant predictors of short PFS(Table 5). For the 3-year OS rates, patients with Slug overexpression represented 34% and patients without,66%, patients with Twist overexpression represented 36% and patients without, 64%, and patients with Snail overexpression represented 18% and patients without, 82%(Table 5). Loss of E-cadherin expression, stage, grade, and CIS were also negative predictors of the OS (Table 5). We failed to demonstrate any significant correlation between OS and Twist, Slug and Snail,(Table 5). Multivariate analysis of prognostic variables in patients with BT In this analysis, we only focused on markers of interest in this study. Doing a multivariate analysis with too many variables, even in 120 patients with BT, is bio-sta- tistical nonsense. As stage, grade, or CIS are well-known prognostic factors in BT, we evaluated the expression of Snail, Slug, Twist and E-cadherin. In multivariate PFS analysis, Snail, Slug, Twist and E-cadherin were entered into the Cox proportional hazard analysis. Only Twist, Slug and E-cadherin expression retained significance as a prognostic factor of a short PFS (OR, 0.276; 95% CI, 0.090-0.841; P = 0.018, OR, 0.656, 95% CI, 0.215-2.003; P = 0.014, and OR, 23.208, 95% CI, 6.113-3.331; P =