Prognostic Impact of Carboxylesterase 2 in Cholangiocarcinoma

Abstract

Carboxylesterase 2 (CES2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. Novel treatment strategies are exceedingly needed for cholangiocarcinoma (CCA) patients. Here, we assessed CES2 expression by immunohistochemistry in a CCA cohort comprising 171 non-liver fluke associated, intrahepatic (n = 72) and extrahepatic (perihilar: n = 56; distal: n = 43) CCAs. Additionally, 80 samples of high-grade biliary intraepithelial neoplastic tissues and 158 corresponding samples of histological normal, non-neoplastic biliary tract tissues were included. CES2 expression was highest in non-neoplastic biliary tissue and significantly decreased in CCA. Patients showing any CES2 expression in tumor cells had a significantly better overall survival compared to negative cases (p = 0.008). This survival benefit was also maintained after stratification of CES2-positive cases, by comparing low, medium and high CES2 expression levels (p-trend = 0.0006). Evaluation of CCA subtypes showed the survival difference to be restricted to extrahepatic tumors. Correlation of CES2 expression with data of tumor-infiltrating immune cells showed that particularly CD8+ T cells were more frequently detected in CES2-positive CCAs. Furthermore, treatment of CCA cell lines with the prodrug Irinotecan reduced cell viability, increased cytotoxicity and modulated inflammatory gene expression. In conclusion, reduced CES2 expression is associated with poor outcome and low CD8+ T cell infiltration in CCA patients. Further clinical studies could show, whether CES2 expression may serve as a predictive marker in patients treated with prodrugs converted by CES2.

Figure 1

CES2 immunohistochemistry of representative CCA cases. Representative TMA core of an iCCA, negative for CES2 by immunohistochemistry (A). TMA core of an iCCA showing homogenous strong cytoplasmic CES2 immunoreactivity, while tumor stroma is CES2 negative (B). TMA core of a pCCA showing homogenous strong cytoplasmic CES2 immunoreactivity (C), while tumor stromal cells are also partly CES2 immunoreactive (D). Original magnification: 40x (A–C), 200x (D).

Figure 2

CES2 immunoreactivity in non-neoplastic normal biliary epithelium, high-grade biliary intraepithelial neoplasias and invasive CCA including subtype-specific evaluation. CES2 immunoreactivity was highest in non-neoplastic, normal biliary epithelium and stepwise decreasing in cholangiocarcinogenesis (A; p < 0.001, p = 0.004 respectively). No significant differences in CES2 immunoreactivity were detected in CCA subtypes (B). P-values were calculated by Mann-Whitney U test. Box plot with 5–95% whiskers.

Figure 3

Overall survival probability in cholangiocarcinoma patients in correlation with CES2 immunoreactivity. Kaplan-Meier curves show a longer overall survival of CCA patients in correlation with higher CES2 immunoreactive scores (A). CCA patients with low (B) or absent (C) CES2 immunoreactivity also showed a significant shortened overall survival compared to patients with higher CES2 immunoreactive scores. P-values were calculated by log-rank test or p-trend (A). Survival data were available for 142 (83%) of 171 CCA patients.

Figure 4

Overall survival probability in cholangiocarcinoma subtypes in correlation with CES2 immunoreactivity. Kaplan-Meier curves show no significant overall survival differences of iCCA (A,B) and pCCA (C,D) patients in correlation with CES2 immunoreactivity, whereas a significant longer overall survival of dCCA patients in correlation with CES2 immunoreactivity was detected (E, p = 0.008; F, p = 0.036). P-values were calculated by log-rank test.

Figure 5

The prodrug Irinotecan reduces cell viability, induces cytotoxicity, and induces the expression of inflammatory genes in CCA cell lines. Cell viability assay of TFK-1 (A) and EGI-1 (B) and cytotoxicity assays of TFK-1 (C) and EGI-1 (D) upon treatment with 20 μg/ml Irinotecan for up to 72 h. Control cells were untreated. Shown are relative mean values with standard deviation (SD) of one out of three independent experiments with similar results. Within each experiment values were normalized to control treated cells. Relative mRNA levels of TFK-1 and EGI-1 cells treated with 20 μg/ml Irinotecan or left untreated for up to 72 h (E,F). Shown are relative mean values with SD of three independent experiments. Within each experiment values were normalized to control treated cells. *Mann-Whitney U test p < 0.05.

Figure 6

Immune cell infiltration in CES2-negative and -positive CCA. Analysis of quantity of CD8+ intraepithelial T cells in correlation with CES2 expression in the CCA cohort (A; Mann Whitney test p = 0.046) and analysis of quantity of total FOXP3+ regulatory T cells in the CCA cohort (B; Mann Whitney test p = 0.046).