Ancers. The diagnostic value of SSP411 as a novel candidate serum diagnostic biomarker for CC was tested using an ELISA assay and receiver operating characteristic (ROC) analysis. Consisted with the proteomic results, CC patients had significantly higher serum levels of SSP411 (mean OD value 6 SD of 0.9260.20) than individualsValidation of PGAM-1, HSPD1, 25033180 PDIA3 and SSP411 expression in surgical tissues by immunoblotting and immunohistochemical analysisWestern blotting was used to quantify the expression of PGAM1, HSPD1, PDIA3 and SSP411 (which were all upregulated in bileFigure 1. 2-DE gels of pooled human bile samples. (A, B) 2-DE maps of pooled bile samples from cholangiocarcinoma patients (A; n = 5 each) and pooled samples from cholangitis patients (B; n = 3 or 4 each). The proteins were separated by 2D-PAGE and visualized by silver staining. The differentially expressed proteins selected as potential biomarkers are marked. doi:10.1371/journal.pone.0047476.gProteomic Study Reveals SSP411 as a CC BiomarkerFigure 2. Validation of the 2-DE results by Western blotting. (A) Left panel: Western blotting analysis of aliquots of pooled bile proteins from patients with CC or cholangitis. Right panel: the corresponding spots, with the same molecular weights, in the 2-DE gels. The Ponceau-S-stained blot (below) was used the internal control. (B) Quantification of protein expression in pooled bile proteins. The bars represent the signal intensity of the immunobands and were consistent with the 2-DE results. doi:10.1371/journal.pone.0047476.gfrom the normal (0.4960.15; P,0.01) and benign groups (0.5960.27; P,0.01, Figure 6A).The ROC area under the curve (AUC) for SSP411 was 0.913 and the Eliglustat custom synthesis Dimethylenastron supplier cut-off point was 0.63, with a sensitivity of 90 and a specificity of 83.3 to distinguish CC from benign disease and normal controls. The AUC was 0.836 and the cut-off point was 0.65 (sensitivity = 85.7; specificity = 76.9) when the CC was compare with the benign group (Figure 6E). Similar to CC, the SSP411 level in HCC patients (0.6460.24) were higher than in the liver cirrhosis (0.4760.16; p,0.05) and normal groups (0.4960.15; p,0.05; Figure 6C). However, the diagnostic efficiency of SSP411 for HCC was significantly lower than for CC (Figure 6D). The sensitivity (HCC vs. cirrhosis, 77.7 ; HCC vs. cirrhosis+ normal, 41.7 ) and specificity (HCC vs. Cirrhosis, 60.0 ; HCC vs. Cirrhosis+ Normal, 84.8 ) of SSP411 for the diagnosis of HCC were not satisfactory (Figure 6, E).DiscussionCholangiocarcinoma (CC) is the second most common primary hepatic malignancy of the biliary-duct system. The typical age of CC is the seventh decade of life, with a slightly higher incidence in men [20]. Our study found an average age of 60.7610.6 yr and male patients were also more likely to be affected than female patients with a ratio of 1.3. Given the poor prognosis 
of CC, mortality and incidence rates are virtually similar. CC incidence rates vary markedly worldwide, which presumably reflects differences in local risk factors and genetic susceptibility. There are a number of established risk factors underlying CC carcinogenesis, such as primary sclerosing cholangitis (PSC), infestation with liver flukes, toxic, biliary-tract disorders, hepatolithiasis, choledocholithiasis and cholangitis, amongst others. However,most patients that present with CC do not have identifiable risk factors [21]. PSC is the most common predisposing factor for CC in the Western countries. This is an autoimm.Ancers. The diagnostic value of SSP411 as a novel candidate serum diagnostic biomarker for CC was tested using an ELISA assay and receiver operating characteristic (ROC) analysis. Consisted with the proteomic results, CC patients had significantly higher serum levels of SSP411 (mean OD value 6 SD of 0.9260.20) than individualsValidation of PGAM-1, HSPD1, 25033180 PDIA3 and SSP411 expression in surgical tissues by immunoblotting and immunohistochemical analysisWestern blotting was used to quantify the expression of PGAM1, HSPD1, PDIA3 and SSP411 (which were all upregulated in bileFigure 1. 2-DE gels of pooled human bile samples. (A, B) 2-DE maps of pooled bile samples from cholangiocarcinoma patients (A; n = 5 each) and pooled samples from cholangitis patients (B; n = 3 or 4 each). The proteins were separated by 2D-PAGE and visualized by silver staining. The differentially expressed proteins selected as potential biomarkers are marked. doi:10.1371/journal.pone.0047476.gProteomic Study Reveals SSP411 as a CC BiomarkerFigure 2. Validation of the 2-DE results by Western blotting. (A) Left panel: Western blotting analysis of aliquots of pooled bile proteins from patients with CC or cholangitis. Right panel: the corresponding spots, with the same molecular weights, in the 2-DE gels. The Ponceau-S-stained blot (below) was used the internal control. (B) Quantification of protein expression in pooled bile proteins. The bars represent the signal intensity of the immunobands and were consistent with the 2-DE results. doi:10.1371/journal.pone.0047476.gfrom the normal (0.4960.15; P,0.01) and benign groups (0.5960.27; P,0.01, Figure 6A).The ROC area under the curve (AUC) for SSP411 was 0.913 and the cut-off point was 0.63, with a sensitivity of 90 and a specificity of 83.3 to distinguish CC from benign disease and normal controls. The AUC was 0.836 and the cut-off point was 
0.65 (sensitivity = 85.7; specificity = 76.9) when the CC was compare with the benign group (Figure 6E). Similar to CC, the SSP411 level in HCC patients (0.6460.24) were higher than in the liver cirrhosis (0.4760.16; p,0.05) and normal groups (0.4960.15; p,0.05; Figure 6C). However, the diagnostic efficiency of SSP411 for HCC was significantly lower than for CC (Figure 6D). The sensitivity (HCC vs. cirrhosis, 77.7 ; HCC vs. cirrhosis+ normal, 41.7 ) and specificity (HCC vs. Cirrhosis, 60.0 ; HCC vs. Cirrhosis+ Normal, 84.8 ) of SSP411 for the diagnosis of HCC were not satisfactory (Figure 6, E).DiscussionCholangiocarcinoma (CC) is the second most common primary hepatic malignancy of the biliary-duct system. The typical age of CC is the seventh decade of life, with a slightly higher incidence in men [20]. Our study found an average age of 60.7610.6 yr and male patients were also more likely to be affected than female patients with a ratio of 1.3. Given the poor prognosis of CC, mortality and incidence rates are virtually similar. CC incidence rates vary markedly worldwide, which presumably reflects differences in local risk factors and genetic susceptibility. There are a number of established risk factors underlying CC carcinogenesis, such as primary sclerosing cholangitis (PSC), infestation with liver flukes, toxic, biliary-tract disorders, hepatolithiasis, choledocholithiasis and cholangitis, amongst others. However,most patients that present with CC do not have identifiable risk factors [21]. PSC is the most common predisposing factor for CC in the Western countries. This is an autoimm.
