Figures & data
Figure 1. Hypermethylated and hypomethylated miRNA promoters in cholangiocarcinoma identified by overlapping genomic DMR and putative miRNA promoter coordinates. A) From 1374 putative miRNA promoter regions,Citation11 27 and 13 overlapped with 1099 hypermethylated and 565 hypomethylated regions, respectively, common to >9/18 CC samples.Citation7 B) Integrated Genomics Viewer (IGV 2.3)Citation41 browser view on an example of a hypermethylated DMR common to 17 of 18 CC patientsCitation7 and overlapping with the promoter of miR-124-3 (see Supplementary Table S2). CGI: CpG island.
Figure 2. Correlation between miRNA promoter methylation and miRNA expression in CC. Methylation levels of the promoters of miR-129-2 (A) and the co-regulated miR-200a, miR-200b, and miR-429 (C) in cohort #2 and control samples. Corresponding relative expression is shown to the right (B and D). In C) and D), additional patient samples were included (see text). NEBD: normal extrahepatic bile duct; NIBD: normal intrahepatic bile duct.
Table 1. Clinicopathological characteristics of cholangiocarcinoma cohorts #1 and #2 used for methylome and miRNA analyses. Cases with pNx had no lymph nodes resected. 16 cases (ID 1-16) were used in our methylome screen.Citation7
Figure 3. CDH6 expression in CC. High immunoreactivity for CDH6 in (A) normal biliary epithelium (large intrahepatic bile ducts) and (D) exemplary in one intrahepatic cholangiocarcinoma. Low immunoreactivity for CDH6 in (B) one extrahepatic cholangiocarcinoma and (C) one intrahepatic cholangiocarcinoma. Original magnification: 50x, 200x (inset).
Figure 4. Statistical analyses of CDH6 expression in BTC and correlation with patient survival. (A) CDH6 immunoreactivity is already significantly reduced in pre-invasive BillN 3 lesions and decreases even more in invasive BTC compared to normal biliary epithelium. (B) Subgroup analysis shows a significant stronger decrease of CDH6 expression for ECC and GBAC compared to ICC (Unpaired non-parametric Mann-Whitney-U test: ** P < 0.005; ns: not significant; Kruskal-Wallis test P < 0.001). (C) Kaplan-Meier curves show that BTC patients with very high CDH6 expression levels (IRS = 12, n = 11 ICCs; see also and Fig. 6D) show significantly prolonged overall survival (red Kaplan-Meier curve) compared to patients with lower CDH6 IRS (blue Kaplan-Meier curve). (D) This also holds true in ICC subtype-specific survival analysis.
Table 2. Clinicopathological characteristics of the biliary tract cancer cohort used for TMA analysis. Cases with pNx had no lymph nodes resected; therefore, UICC status could not be assessed precisely in these cases. ‡Mann Whitney U-test; ‡‡Fisher's exact test; ‡‡‡Chi-square test.
Figure 5. Growth curves of the CC cell lines EGI-1 (A) and TFK-1 (B) after lentiviral transfection with CDH6 cDNA or GFP. Initial cell number of EGI-1 was 2,500, that of TFK-1 was 313. Relative fluorescence units (RU) were determined after 24, 48, 72, and 96 h. Values are means of 3 technical replicates; error bars indicate standard deviation. untr.: untransfected.
