Wang et al. Journal of Experimental & Clinical Cancer Research 2012, 31:44 http://www.jeccr.com/content/31/1/44 Page 4 of 7 corresponding to the glioma WHO grades. The statistic results (Figure 1B) showed that its expression in high- grade (III-IV; 2.2±0.08) and low-grade (I-II; 1.6±0.06) gliomas were both significantly higher than that in non- neoplastic brains tissues (0.3±0.01; both P<0.001). Add- itionally, there was also a significant difference in mRNA copies of CLIC1 between high-grade (III-IV) and low -grade (I-II) glioma tissue specimens (P=0.002). Elevated expression of CLIC1 protein in human glioma tissues The expression of CLIC1 protein was detected in 128 glioma specimens and 10 nonneoplastic brain tissues using immunohistochemical staining. Representative photographs of CLIC1 immunostainings were shown in Figure 2. In the glioma sections, CLIC1 was mainly detected in the cytoplasm (Figure 2A), which was con- sistent with previous studies on other cancers [10-12]. In contrast, the non-neoplastic brain tissues expressed a trace amount of CLIC1 (Figure 2B). CLIC1 was not present in negative controls with non-immune IgG (Figure 2C) and in normal gastric tissues (Figure 2D). Of the 128 patients with gliomas, the high expression of CLIC1 was detected in 69.5% (89/128) of patients. For WHO grade III and IV tumors, 79.2% (76/96) of cases highly expressed CLIC1. However, for grade I and grade Figure 1 CLIC1 mRNA expression in 20 glioma tissues with different grades and in non-neoplastic brain tissues were detected by real-time quantitative RT-PCR assay. (A) Expression levels of CLIC1 mRNA in glioma tissues with different grades and non-neoplastic brain tissues. (B) A graphical representation of the CLIC1 mRNA level expression profiles in (A). ‘N’ refers to non- neoplastic brain tissues; ‘I~II’ refers to glioma tissues with grade I~II; ‘III~IV’ refers to glioma tissues with grade III~IV. II tumors, 40.6% (13/32) of cases highly expressed CLIC1. According to these results, increased expression of CLIC1 was found to be associated with the histo- pathologic grading of the gliomas. Association of CLIC1 expression with clinicopatholigcal features of gliomas The associations of CLIC1 protein expression with the clinicopathological factors of the glioma patients were summarized in Table 2. The over-expression of CLIC1 was detected in high-grade glioma tissues compared with those in low-grade tissues, and increased with ascending tumor WHO grades (P=0.005, Table 2). The increased expression of CLIC1 protein was also signifi- cantly correlated with low Karnofsky performance score (KPS) (P=0.008, Table 2). No statistically significant asso- ciations of CLIC1 with age at diagnosis and gender of patients were found (both P>0.05, Table 2). Association of CLIC1 expression with overall survival in patients with gliomas Kaplan-Meier analysis using the log-rank test was per- formed to determine the association of CLIC1 expression with clinical outcome of glioma patients (Figure 3A). The results shown that high expression of CLIC1 was mark- edly associated with a shorter overall survival (P<0.001). During the follow-up period, 100 of 128 glioma patients (78.1%) had died. Of patients with high CLIC1 expression, 81 (81/89, 91.0%) were died; in contrast, 19 (19/39, 48.7%) of patients with low CLIC1 expression were died. The me- dian survival time of patients with high CLIC1 expression (28.6 months, 95% confidence interval: 25.6–33.9) was sig- nificantly shorter than that of patients who had low CLIC1 expression level (50.1 months, 95% confidence interval: 41.2–58.6, P<0.001). Nextly, the univariate analysis of individual variables revealed strong relationships between overall survival and WHO grade (P< 0.001), and CLIC1 expression (P<0.001). Additionally, the multivariate analysis identified CLIC1 ex- pression (HR, 4.66; 95% CI, 2.31–10.29; P=0.01) and WHO grade (HR, 6.97; 95% CI, 2.12–12.46; P=0.008) as significant prognostic factors for glioma (Table 3). Furthermore, we evaluated the prognostic significance of CLIC1 protein expression levels in different sub- groups of glioma patients stratified according to the WHO grading. Notably, high CLIC1 expression also sig- nificantly correlated with shorter overall survival time in different glioma subgroups. Overall survival of glioma patients with high CLIC1 expression was significantly decreased than those with low CLIC1 expression in ei- ther Grades I~II subgroup (n=32; P=0.01; Figure 3B) or Grades III~IV subgroup (n=96; P=0.008; Figure 3C).