Cholangiocarcinoma (CCA) is an aggressive tumor of the bile duct with a high rate of mortality. Lymph node metastasis is an important factor facilitating the progression of CCA. A reliable biomarker for diagnosis, progression status, or prognosis of CCA is still lacking. To identify a novel and reliable biomarker for diagnosis/prognosis of CCA, liquid chromatography-mass spectrometry and tandem mass spectrometry (LC-MS/MS) in combination with bioinformatics analysis were applied for the representative serum samples of patients with CCA. The proteome results showed that protein tyrosine phosphatase receptor S (PTPRS) had the highest potential candidate. Then, a dot blot assay was used to measure the level of serum PTPRS in patients with CCA (n = 80), benign biliary disease patients (BBD; n = 39), and healthy controls (HC; n = 55). PTPRS level of CCA sera (14.38 ± 9.42 ng/ml) was significantly higher than that of BBD (10.7 ± 5.05 ng/ml) or HC (6 ± 3.73 ng/ml) (P < 0.0001). PTPRS was associated with serum albumin (P = 0.028), lymph node metastasis (P = 0.038), and the survival time of patients (P = 0.011). Using a log-rank test, higher serum PTPRS level was significantly (P = 0.031) correlated with a longer overall survival time of patients with CCA, and PTPRS was an independent prognostic marker for CCA superior to carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA) or alkaline phosphatase (ALP). High expression of PTPRS could be a good independent prognostic marker for CCA.
Distal cholangiocarcinoma (dCCA) is a rare type of CCA in Asia, even in Opisthorchis viverrini-prevalent Northeastern Thailand. The clinical ambiguity and imprecision of diagnosis surrounding this malignancy result in high mortality due often to advanced/metastatic disease on presentation. We aim to identify a prognostic factor that can improve the performance stratification and influence the outcome of dCCA patients after curative resection. A total of 79 patients who underwent curative-intended surgery for dCCA was enrolled. Possible risk factors for survival were analyzed with log-rank test, and independent factors with Cox regression model. dCCA patients were staged and classified according to the 8th edition the American Joint Committee on Cancer (AJCC) Staging Manual. Results were then compared with the revised classification employing the prognostic factor identified from multivariate analysis. Multivariate analysis revealed that growth pattern (p < 0.01) and distant metastasis (p = 0.012) were independent factors. Growth patterns comprise intraductal (ID), periductal infiltrating (PI), mass-forming (MF), and mixed types. When dCCA patients were grouped into those having good and poor outcomes (with and without ID components, respectively). The survival outcomes significantly differed among patients with and without ID components, which was better than with the 8th AJCC staging system in our cohort. Furthermore, Chi-square test showed that patterns without ID components (PI, MF, PI + MF) correlated with lymph node and distant metastasis. Therefore, classification of dCCA patients after curative-intended surgical resection based on growth pattern provides additional beneficial information for the prediction of survival in dCCA patients.
Cholangiocarcinoma (CCA) is a tumor arising from cholangiocytes lining the bile ducts. Vascular invasion and lymph node metastasis are important prognostic factors for disease staging as well as clinical therapeutic decisions for CCA patients. In the present study, we applied CCA sera proteomic analysis to identify a potential biomarker for prognosis of CCA patients. Then, using bioinformatics tools, we identified angiopoietin-like protein 4 (ANGPTL4) which expressed highest signal intensity among candidate proteins in proteomic analysis of CCA sera. Expression of ANGPTL4 in CCA tissues was determined using immunohistochemistry. The results showed that ANGPTL4 was stained at higher level in CCA cells when compared with normal cholangiocytes. The high expression of ANGPTL4 was associated with lymph node metastasis and advanced tumor stage (p = 0.013 and p = 0.031, respectively). Furthermore, serum ANGPTL4 levels in CCA and healthy control (HC) were analyzed using a dot blot assay. And it was found that ANGPTL4 level was significantly higher in CCA than HC group (p < 0.0001). ROC curve analysis revealed that serum ANGPTL4 level was effectively distinguished CCA from healthy patients (cutoff = 0.2697 arbitrary unit (AU), 80.0% sensitivity, 72.7% specificity, AUC = 0.825, p < 0.0001). Serum ANGPTL4 level was associated with vascular invasion and lymph node metastasis (p = 0.0004 and p = 0.006), so that it differentiated CCA with vascular invasion from CCA without vascular invasion (cutoff = 0.5526 AU, 64.9% sensitivity, 92.9% specificity, AUC = 0.751, p = 0.006) and it corresponded to CCA with/without lymph node metastasis (cutoff = 0.5399 AU, 71.4% sensitivity, 70.8% specificity, AUC = 0.691, p = 0.01) by ROC analysis. Serum ANGPTL4 levels showed superior predictive efficiency compared with CA 19-9 and CEA for vascular invasion and lymph node metastasis. In addition, serum ANGPTL4 level was an independent predictive indicator by multivariate regression analysis. In conclusion, serum ANGPTL4 could be a novel prognostic biomarker for prediction of vascular invasion and lymph node metastasis of CCA patients.
Gemcitabine and cisplatin serve as appropriate treatments for patients with cholangiocarcinoma (CCA). Our previous study using histoculture drug response assay (HDRA), demonstrated individual response patterns to gemcitabine and cisplatin. The current study aimed to identify predictive biomarkers for gemcitabine and cisplatin sensitivity in tissues and sera from patients with CCA using metabolomics. Metabolic signatures of patients with CCA were correlated with their HDRA response patterns. The tissue metabolic signatures of patients with CCA revealed the inversion of the TCA cycle that is evident with increased levels of citrate and amino acid backbones as TCA cycle intermediates, and glucose which corresponds to cancer stem cell (CSC) properties. The protein expression levels of CSC markers were examined on tissues and showed the significantly inverse association with the responses of patients to cisplatin. Moreover, the elevation of ethanol level was observed in gemcitabine- and cisplatin-sensitive group. In serum, a lower level of glucose but a higher level of methylguanidine was observed in the gemcitabine-responders as non-invasive predictive biomarker for gemcitabine sensitivity. Collectively, our findings indicate that these metabolites may serve as the predictive biomarkers in clinical practice which not only predict the chemotherapy response in patients with CCA but also minimize the adverse effect from chemotherapy.
Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent and patented recently as a potential chemotherapeutic drug for various cancers. The aims of this study were, therefore, to investigate the ability of PP in anti-proliferative activity and focused on the lipid profiles revealing the alteration of specific lipid species in the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability through suppressing mitochondrial membrane potential (MMP) and ATP productions, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with chemometrics was performed to investigate lipid alteration during PP-induced apoptosis. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate, and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. The orthogonal partial-least squares regression analysis revealed that PP-induced MMP dysfunction, leading to remarkably reduced ATP level, was significantly associated with triglyceride (TG) accumulation observed in PP-treated CCA cells. Our findings indicate that PP could suppress the MMP function, which causes inhibition of CCA cell viability through lipid production, resulting in apoptotic induction in CCA cells. These findings provide an anti-cancer mechanism of PP under apoptotic induction ability that may serve as the alternative approach for CCA treatment.