Fan et al. BMC Cancer 2011, 11:320 http://www.biomedcentral.com/1471-2407/11/320 expression of CD133 was significantly correlated with well or moderately differentiated CC (P = 0.004, Table 2), which implied that the positive expression meant higher differentiation status of tumors while the absence of CD133 signified poorly differentiated ones. Among the 40 cases of CD133-positive expression, the well-differentiated tumors were stained mostly on the membrane or on the both membrane and in the cyto- plasm (17 of 19, 89%) but the majority of moderately and poorly differentiated tumors were stained in the cytoplasm or both on the membrane and in the cyto- plasm (8/10, 80% and 10/11, 91%, respectively); more- over, by statistical anlysis, the different subcellular localization of CD133 correlated with the differentiation status of the tumor (P = 0.009, Table 2). However, the age and gender of the patients did not correlate with the CD133 expression or the differentiation status of CC (P >0.05). CD133 Negative Expression in CC Predicts Worse Outcome Twenty-three patients were followed up for survival. Among them, 14 cases were CD133 positive and the other 9 cases were CD133 negative in their tumors. Using Kaplan-Meier analysis, the median survival was 4 months for CD133 negative patients and 14 months for the positive ones. Log-rank test showed that the CD133 positive patients had a significantly better survival than negative ones (P = 0.001) and the negative expression of CD133 was a worse prognostic indicator for patients with CC (Figure 3). The univariate analysis indicated that the differentiation status (P = 0.025), staging (P = 0.006) and gender (P = 0.005) were also significant prog- nostic indicators, with the less well differentiated tumor and male gender predicating for poor overall survival for patients with CC (Table 3). To clarify the indepen- dent prognostic value of the CD133 expression in the patients with CC, a multivariate analysis of the relevant parameters was performed. The COX regression analysis revealed that CD133 expression (P = 0.007) and the Table 2 CD133 expression in CC CC Number of cases CD133 negative CD133 positive Membrane Membrane & Cytoplasm Cytoplasm 21 2 19 (90%) 8 9 2 * P = 0.004 (CD133 positivity correlates with differentiation status); # P = 0.009 (CD133 subcelluar localization correlates with differentiation status) P < 0.05 is considered significant Well differentiated Moderately differentiated Poorly differentiated Page 4 of 8 Figure 3 CD133 negative expression correlates with poor survival in CC. Kaplan-Meier survival curve showed the association between CD133 positive and negative expression in 23 patients with CC. Fourteen patients with positive CD133 expression had mean overall survival of 14 months versus 4 months for nine patients with CD133 negative expression. The log-rank test demonstrated CD133 negative expression correlated with poor outcome in CC (P = 0.001). differentiation status (P = 0.010) were prognostic factors for overall survival (Table 3). Discussion In this study, we investigated CD133 expression in nor- mal human liver tissues and CC tissues using the immu- nohistochemical method. We found that CD133 was expressed in human normal livers but the expression was restricted to biliary trees on the apical membrane. In CC, 40 of 54 (74%) cases studied showed CD133 expression with different degrees. Similar to colon and mammary gland adenocarcinoma which have high expression of CD133 in well or moderately differentiated tumors [27], most of the well or moderately differen- tiated CC (90% and 83%, respectively) expressed CD133 and moreover, most CD133 negative CC tumors (71%) were poorly differentiated. 12 2 10 (83%) 2 4 4 21 10 11 (52%) 1 5 5 Subtotal 54 14 40 (74%) *# 11 18 11