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Abstract Background The Cholangiocarcinoma Screening and Care Program (CASCAP) has been launched since 2013 to detect early-stage cholangiocarcinoma and reduce the disease death. However, the clinical utility of the CASCAP remains unclear. To compare survival outcomes between two time periods: before and after 2013, when significant changes in treatment strategies were implemented, and to evaluate the efficacy of the ultrasound-based screening program, in an Asian region endemic for cholangiocarcinoma. Methods A single-center retrospective review was conducted in curative-intended resection cholangiocarcinoma from 2002 to 2021. Patents characteristics and survival outcomes were compared between 2002 and 2013 (early period) and 2014 to 2021 (later period). Results A total of 1091 patients with intrahepatic (n = 624) or perihilar (n = 467) cholangiocarcinoma was included (early period, n = 658; later period, n = 433). Of 66 (15.2%) patients in the later period were referred by the CASCAP. The incidence of early-staged disease (Stage 0 and 1) was lower in early period compared to later period 16.0% versus 29.1% (p

Background: Most cholangiocarcinoma (CCA) patients present with late stage of disease because of the difficulty to diagnosis at an early stage, resulting in poor survival of CCA patients. The Cholangiocarcinoma Screening and Care Program showed that ultrasound screening was an effective tool for detecting early stage CCA. This study aims to evaluate the survival outcome of patients diagnosed by ultrasound screening (US) compared to walk-in symptomatic patients. Methods: The retrospective clinical data and medical records for this study were accessed in June 30, 2021. 5-year survival rates (5-YSR) and median survival time (MST) of CCA were calculated using the Kaplan-Meier method. Multivariate analyses were performed for significant factors from univariate analyses. Results: A total of 711 histologically proven CCA cases were examined including ultrasound screening and walk-in groups. The screening group having 5-YSR was 53.9 %, and MST was of 67.2 months, while walk-in group, the 5-YSR was 21.9 % and MST was 15.6 months (p

Background: The epithelial malignant tumor known as cholangiocarcinoma (CCA) is most commonly found in Southeast Asia, particularly in northeastern Thailand. Previous research has indicated that the overexpression of acyl-CoA synthetase long-chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11), and ChaC glutathione-specific γ-glutamylcyclotransferase (CHAC1) as ferroptosis-related proteins is associated with poorer prognosis in several cancers. The role of these three proteins in CCA is still unclear. The present study aimed to investigate the expression levels of ACSL4, SLC7A11, and CHAC1, all potential ferroptosis biomarkers, in CCA. Methods: The ACSL4, SLC7A11, and CHAC1 protein expression levels in 137 CCA tissues were examined using immunohistochemistry, while 61 CCA serum samples were evaluated using indirect ELISA. The associations between the expression levels of ACSL4, SLC7A11, and CHAC1 and patient clinicopathological data were evaluated to determine the clinical significance of these proteins. Results: The expression levels of ACSL4, SLC7A11, and CHAC1 were assessed in CCA tissues. A significant association was observed between high ACSL4 levels and extrahepatic CCA, tumor growth type, and elevated alanine transferase (ALT). There was also a positive association between elevated SLC7A11 levels and tumor growth type. Additionally, the upregulation of CHAC1 was significantly associated with a shorter survival time in patients. High levels of ACSL4 and SLC7A11 in CCA sera were both significantly associated with advanced tumor stages and abnormal liver function test results, indicating that they could be used as a reliable prognostic biomarker panel in patients with CCA. Conclusions: The results of the present study demonstrated that the upregulation of ACSL4, SLC7A11, and CHAC1 could be used as a valuable biomarker panel for predicting prognosis parameters in CCA. Furthermore, ACSL4 and SLC7A11 could potentially serve as complementary markers for improving the accuracy of prognosis prediction when CCA sera is used. These less invasive biomarkers could facilitate effective treatment planning.

Objective: To study which radiographic features were associated with recurrence and adverse outcome in patients undergoing surgical resection of perihilar cholangiocarcinoma (PCCA), as well as to evaluate the imaging patterns that signify recurrence after the resection of PCCA. Materials and methods: This study was conducted in a solitary tertiary center and utilized a retrospective, analytical, case-control design. The study population consisted of patients with pathologically confirmed PCCA who underwent surgical resection and were subsequently followed up from January 2009 to December 2017. A total of 77 patients were enrolled in the study and were categorized into two distinct groups, namely recurrent and non-recurrent. The analysis encompassed the examination of demographic data and recurrence patterns. Additionally, survival and multivariate analyses were employed to assess radiographic imaging data and surgical information. Results: Seventy-seven patients diagnosed with PCCA based on pathological evidence were included in the study. Among the participants, there were 28 females and 49 males, with ages ranging from 41 to 81 years (mean age of 60.65 ± 7.66). A noteworthy finding was the recurrence rate of 65 % observed following surgical resection. The presence of regional lymph node (LN) metastasis, adjacent organ invasion, and surgical margin emerged as the three independent factors that exhibited a significant association with recurrence after post-operative resection (p = 0.023, p = 0.028, and p = 0.010, respectively). The patients with PCCA who experienced regional LN metastasis had a median overall survival (OS) of 22 months, which was significantly lower than the 46 months observed in those without regional LN metastasis (p

Intraductal T2 mapping based on a catheter receiver is proposed as a method of visualizing the extent of intraductal and periductal cholangiocarcinoma (CCA). Compared to external receivers, internal receivers provide locally enhanced signal-to-noise ratios by virtue of their lower field-of-view for body noise, allowing smaller voxels and higher resolution. However, inherent radial sensitivity variation and segmentation for patient safety both distort image brightness. We discuss simulated T2 weighted images and T2 maps, and in vitro images obtained using a thin film catheter receiver of a freshly resected liver specimen containing a polypoid intraductal tumor from a patient with CCA. T2 mapping provides a simple method of compensating non-uniform signal reception patterns of catheter receivers, allowing the visualization of tumor extent without contrast enhancement and potentially quantitative tissue characterization. Potential advantages and disadvantages of in vivo intraductal imaging are considered.

Abstract Background Screening for opisthorchiasis, a parasitic worm infection affecting many millions of people in Southeast Asia, has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique (FECT) and Kato-Katz method. Although the urinary enzyme-linked immunosorbent assay (ELISA) has been used more recently, we developed a urinary antigen-based rapid diagnostic test (RDT) to simplify diagnosis and as a point-of-care testing (POCT) and field applications for surveillance and control of opisthorchiasis. Methods A urinary Opisthorchis viverrini (OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV. The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA (n = 493). Cross-reactivities of urinary OV-RDT with other helminthiases coexisted with O. viverrini were determined (n = 96). A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis (n = 1629). The McNemar chi-square, Kruskal–Wallis and Cohen’s kappa coefficient (κ-value) tests were used for statistical analyses. Results Urinary OV-RDT had sensitivity of 94.2% and specificity of 93.2%, compared to faecal FECT. Urinary OV-RDT had high diagnostic agreement (Kappa = 0.842–0.874, P 0.8, P 0.05). Conclusions The urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis. The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening, control and elimination of opisthorchiasis, thereby contributing to a reduction in the disease burden in Southeast Asia. Graphical Abstract

Background Niclosamide is an oral anthelminthic drug that has been used for treating tapeworm infections. Its mechanism involves the disturbance of mitochondrial membrane potential that in turn inhibits oxidative phosphorylation leading to ATP depletion. To date, niclosamide has been validated as the potent anti-cancer agent against several cancers. However, the molecular mechanisms underlying the effects of niclosamide on the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cell functions remain to be elucidated. The aims of this study were to investigate the effects of niclosamide on CCA cell proliferation and on metabolic phenoconversion through the alteration of metabolites associated with mitochondrial function in CCA cell lines. Materials and Methods The inhibitory effect of niclosamide on CCA cells was determined using SRB assay. A mitochondrial membrane potential using tetramethylrhodamine, ethyl ester-mitochondrial membrane potential (TMRE-MMP) assay was conducted. Liquid chromatography-mass spectrometry-based metabolomics was employed to investigate the global metabolic changes upon niclosamide treatment. ATP levels were measured using CellTiter-Glo® luminescent cell viability assay. NAD metabolism was examined by the NAD+/NADH ratio. Results Niclosamide strongly inhibited CCA cell growth and reduced the MMP of CCA cells. An orthogonal partial-least square regression analysis revealed that the effects of niclosamide on suppressing cell viability and MMP of CCA cells were significantly associated with an increase in niacinamide, a precursor in NAD synthesis that may disrupt the electron transport system leading to suppression of NAD+/NADH ratio and ATP depletion. Conclusion Our findings unravel the mode of action of niclosamide in the energy depletion that could potentially serve as the promising therapeutic strategy for CCA treatment.

Background: Nutritional status is one of the important factors determining the short- and long-term outcomes of surgery in cancer. This study aimed to assess the prognostic role of preoperative controlling nutritional status (CONUT) score in intrahepatic cholangiocarcinoma (iCCA) patients. Methods: A total of 101 iCCA patients who underwent hepatectomy between 2015 and 2018 at the Srinagarind Hospital, Khon Kaen University, were included in this retrospective study. Patients were classified according to the CONUT score. Univariate and multivariate analyses were performed to determine the correlation between clinicopathological features and overall survival. Results: Patients were categorized into normal nutrition (n = 40 or 39.5%), mild (n = 54 or 53.5%), and moderate-severe malnutrition (n = 7). Patients with high CONUT scores had significantly shorter survival (HR 2.55, 95% CI 1.04–6.25, p = 0.04). In multivariable analysis, tumor size (HR = 2.58, p

Abstract The three-dimensional multicellular spheroid (3D MCS) model has been employed in cholangiocarcinoma research as it generates 3D architecture and includes more physiological relevance with the multicellular arrangement. However, it is also essential to explain the molecular signature in this microenvironment and its structural complexity. The results indicated that poorly differentiated CCA cell lines were unable to form 3D MCS due to the lack of cell adhesion molecules with more mesenchymal marker expression. The well-differentiated CCA and cholangiocyte cell lines were able to develop 3D MCSs with round shapes, smooth perimeter, and cell adhesion molecules that led to the hypoxic and oxidative microenvironment detected. For MMNK-1, KKU-213C, and KKU-213A MCSs, the proteo-metabolomic analysis showed proteins and metabolic products altered compared to 2D cultures, including cell–cell adhesion molecules, energy metabolism-related enzymes and metabolites, and oxidative-related metabolites. Therefore, the 3D MCSs provide different physiological states with different phenotypic signatures compared to 2D cultures. Considering the 3D model mimics more physiological relevance, it might lead to an alternate biochemical pathway, targeting to improve drug sensitivity for CCA treatment.

Background: Cholangiocarcinoma (CCA) is a potentially lethal disease that requires surgical treatment. There are many benign conditions, those that do not require surgical treatment, displaying imaging characteristics that resemble CCA, leading to therapeutic misadventures. This study aimed to evaluate the proportion of benign conditions that underwent hepatic resection for presumed CCA, and explore the differences between the characteristics of these two entities stratified by primary imaging features of the lesions. Methods: This retrospective study ran between January 2004 and December 2011. We reviewed the pathological records of all 1,402 patients who underwent curative-intent hepatic resection for preoperatively diagnosed CCA. All clinical and pathological parameters were analyzed. Results: The proportion of benign conditions that underwent hepatic resection was 3.78% (53/1,402). For mass-forming lesions, CCA was associated with higher age, alkaline phosphatase, tumor markers, neutrophil to lymphocyte ratio, and mass size. The patients with mass-forming benign conditions were more associated with diabetes mellitus. For lesions with bile duct dilatation, intraductal tumor, including CCA, had higher CA19-9 level and NLR with statistical significance. For cystic lesions, biliary cystic neoplasm had statistical significance in the rate of abdominal pain. Conclusions: The proportion of hepatic resection for benign conditions was quite low. There were many characteristic differences used to differentiate benign mass-forming conditions from CCA, whereas only few markers in the setting of cystic lesions and biliary dilatation. We recommend performing standard hepatic resection in every case, regardless of the type of imaging features, if the lesion has a chance of being CCA.

Background The liver fluke Opisthorchis viverrini (OV), which subsequently inhabits the biliary system and results in periductal fibrosis (PDF), is one of the primarily causes of cholangiocarcinoma (CCA), a bile duct cancer with an exceptionally high incidence in the northeast of Thailand and other Greater Mekong Subregion (GMS) countries. Insights in fecal metabolic changes associated with PDF and CCA are required for further molecular research related to gut health and potential diagnostic biological marker development. Methods In this study, nuclear magnetic resonance (NMR) metabolomics was applied for fecal metabolic phenotyping from 55 fecal water samples across different study groups including normal bile duct, PDF and CCA groups. Results By using NMR spectroscopy-based metabolomics, fecal metabolic profiles of patients with CCA or PDF and of individuals with normal bile duct have been established with a total of 40 identified metabolites. Further multivariate statistical analysis and hierarchical clustering heat map have demonstrated the PDF- and CCA-specific metabotypes through various altered metabolite groups including amino acids, alcohols, amines, anaerobic glycolytic metabolites, fatty acids, microbial metabolites, sugar, TCA cycle intermediates, tryptophan catabolism substrates, and pyrimidine metabolites. Compared to the normal bile duct group, PDF individuals showed the significantly elevated relative concentrations of fecal ethanol, glycine, tyrosine, and N-acetylglucosamine whereas CCA patients exhibited the remarkable fecal metabolic changes that can be evident through the increased relative concentrations of fecal uracil, succinate, and 5-aminopentanoate. The prominent fecal metabolic alterations between CCA and PDF were displayed by the reduction of relative concentration of methanol observed in CCA. The metabolic alterations associated with PDF and CCA progression have been proposed with the involvement of various metabolic pathways including TCA cycle, ethanol biogenesis, hexamine pathway, methanol biogenesis, pyrimidine metabolism, and lysine metabolism. Among them, ethanol, methanol, and lysine metabolism strongly reflect the association of gut-microbial host metabolic crosstalk in PDF and/or CCA patients. Conclusion The PDF- and CCA-associated metabotypes have been investigated displaying their distinct fecal metabolic patterns compared to that of normal bile duct group. Our study also demonstrated that the perturbation in co-metabolism of host and gut bacteria has been involved from the early step since OV infection to CCA tumorigenesis.

Patients with distal cholangiocarcinoma (dCCA) generally have poor outcomes because of late presentation and diagnosis. Therefore, prognostic factors for predicting outcomes are essential to improve therapeutic strategies and quality of life. Tumor-infiltrating lymphocytes (TILs) have been reported as a prognostic predictor in several cancers. However, their role in dCCA is still unclear. This study aimed to evaluate the association of TILs with outcome in patients with dCCA. Fifty-two patients were evaluated for the percentage rate of TILs in their cancers, and a median TIL level was used to divide the patients into two groups. Survival, multivariate, and correlation analyses were performed to determine the prognostic factors. Results showed that a low TIL level was associated with poor survival. Multivariate analysis revealed TILs as an independent factor for poor outcome. Moreover, TILs were markedly correlated with growth patterns, and both were applied to classify patients with dCCA. Subgroups of TILs with growth pattern incorporation improved stratification performance in separating good from poor patient outcomes. This study suggested that TILs could be a prognostic factor for predicting survival and for clustering patients with dCCA to improve prognostication capability. This finding may be incorporated into a new staging system for stratifying dCCA in Thailand.

AimThis study aims to improve the classification performance of the eighth American Joint Committee on Cancer (AJCC) staging system for perihilar cholangiocarcinoma (pCCA) by proposing the Khon Kaen University (KKU) staging system developed in cholangiocarcinoma-prevalent Northeast Thailand.MethodFour hundred eighty-eight patients with pCCA who underwent partial hepatectomy between 2002 and 2017 at the Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand, were included. Overall survival (OS) related to clinicopathological features was analyzed using the Kaplan–Meier method. Logrank test was performed in univariate analysis to compare OS data of clinicopathological features to determine risk factors for poor survival. Significant features were further analyzed by multivariate analysis (Cox regression) to identify prognostic factors which were then employed to modify the eighth AJCC staging system.ResultsMultivariate analysis showed that growth pattern (HR = 4.67–19.72, p < 0.001), moderately and poorly differentiated histological grades (HR = 2.31–4.99, p < 0.05 and 0.001, respectively), lymph node metastasis N1 and N2 (HR = 1.37 and 2.18, p < 0.05 and 0.01, respectively), and distant metastasis (HR = 2.11, p < 0.001) were independent factors when compared to their respective reference groups. There was a clear separation of patients with pCCA into KKU stage: I [OS = 116 months (mo.)], II (OS = 46 mo.), IIIA (OS = 24 mo.), IIIB (11 mo.), IVA (OS = 7 mo.), and IVB (OS = 6 mo.).ConclusionThe new staging system was based on the incorporation of growth patterns to modify the eighth AJCC staging system. The classification performance demonstrated that the KKU staging system was able to classify and distinctly separate patients with pCCA into those with good and poor outcomes. It was also able to improve the stratification performance and discriminative ability of different stages of pCCA classification better than the eighth AJCC staging system. Hence, the KKU staging system is proposed as an alternative model to augment the accuracy of survival prognostication and treatment performance for patients with pCCA.

Abstract Background Cholangiocarcioma (CCA) treatment is challenging because most of the patients are diagnosed when the disease is advanced, and cancer recurrence is the main problem after treatment, leading to low survival rates. Therefore, our understanding of the mechanism underlying CCA recurrence is essential in order to prevent CCA recurrence and improve patient outcomes. Methods We performed 1H-NMR and UPLC-MS-based metabolomics on the CCA serum. The differential metabolites were further analyzed using pathway analysis and potential biomarker identification. Results At an early stage, the metabolites involved in energy metabolisms, such as pyruvate metabolism, and the TCA cycle, are downregulated, while most lipids, including TGs, PCs, PEs, and PAs, are upregulated in recurrence patients. This metabolic feature has been described in cancer stem-like cell (CSC) metabolism. In addition, the CSC markers CD44v6 and CD44v8-10 are associated with CD36 (a protein involved in lipid uptake) as well as with recurrence-free survival. We also found that citrate, sarcosine, succinate, creatine, creatinine and pyruvate, and TGs have good predictive values for CCA recurrence. Conclusion Our study demonstrates the possible molecular mechanisms underlying CCA recurrence, and these may associate with the existence of CSCs. The metabolic change involved in the recurrence pathway might be used to determine biomarkers for predicting CCA recurrence.

Background Cholangiocarcinoma (CCA) is a malignancy of the cholangiocytes. One of the major issues regarding treatment for CCA patients is the development of chemotherapeutic resistance. Recently, the association of intratumoral bacteria with chemotherapeutic response has been reported in many cancer types. Method In the present study, we aimed to investigate the association between the intratumoral microbiome and its function on gemcitabine and cisplatin response in CCA tissues using 16S rRNA sequencing and 1H NMR spectroscopic analysis. Result The results of 16S rRNA sequencing demonstrated that Gammaproteobacteria were significantly higher in both gemcitabine- and cisplatin-resistance groups compared to sensitive groups. In addition, intratumoral microbial diversity and abundance were significantly different compared between gemcitabine-resistant and sensitive groups. Furthermore, the metabolic phenotype of the low dose gemcitabine-resistant group significantly differed from that of low dose gemcitabine-sensitive group. Increased levels of acetylcholine, adenine, carnitine and inosine were observed in the low dose gemcitabine-resistant group, while the levels of acetylcholine, alpha-D-glucose and carnitine increased in the low dose cisplatin-resistant group. We further performed the intergrative microbiome-metabolome analysis and revealed a correlation between the intratumoral bacterial and metabolic profiles which reflect the chemotherapeutics resistance pattern in CCA patients. Conclusion Our results demonstrated insights into the disruption of the microbiome and metabolome in the progression of chemotherapeutic resistance. The altered microbiome-metabolome fingerprints could be used as predictive markers for drug responses potentially resulting in the development of an appropriate chemotherapeutic drug treatment plan for individual CCA patients.

Cancer-associated fibroblasts (CAFs) are the dominant component of the tumor microenvironment (TME) that can be beneficial to the generation and progression of cancer cells leading to chemotherapeutic failure via several mechanisms. Nevertheless, the roles of CAFs on anti-cancer drug response need more empirical evidence in cholangiocarcinoma (CCA). Herein, we examined the oncogenic roles of CAFs on gemcitabine resistance in CCA cells mediated via IL-6/STAT3 activation. Our findings showed that CCA-derived CAFs promote cell viability and enhance gemcitabine resistance in CCA cells through the activation of IL-6/STAT3 signaling. High expression of IL-6R was correlated with a poor overall survival rate and gemcitabine resistance in CCA, indicating that IL-6R can be a prognostic or predictive biomarker for the chemotherapeutic response of CCA patients. Blockade of IL-6R on CCA cells by tocilizumab, an IL-6R humanized antihuman monoclonal antibody, contributed to inhibition of the CAF-CCA interaction leading to enhancement of gemcitabine sensitivity in CCA cells. The results of this study should be helpful for modifying therapeutic regimens aimed at targeting CAF interacting with cancer cells resulting in the suppression of the tumor progression but enhancement of drug sensitivity.

Abstract Background Sulfasalazine (SSZ) is widely known as an xCT inhibitor suppressing CD44v9-expressed cancer stem-like cells (CSCs) being related to redox regulation. Cholangiocarcinoma (CCA) has a high recurrence rate and no effective chemotherapy. A recent report revealed high levels of CD44v9-positive cells in CCA patients. Therefore, a combination of drugs could prove a suitable strategy for CCA treatment via individual metabolic profiling. Methods We examined the effect of xCT-targeted CD44v9-CSCs using sulfasalazine combined with cisplatin (CIS) or gemcitabine in CCA in vitro and in vivo models and did NMR-based metabolomics analysis of xenograft mice tumor tissues. Results Our findings suggest that combined SSZ and CIS leads to a higher inhibition of cell proliferation and induction of cell death than CIS alone in both in vitro and in vivo models. Xenograft mice showed that the CD44v9-CSC marker and CK-19-CCA proliferative marker were reduced in the combination treatment. Interestingly, different metabolic signatures and significant metabolites were observed in the drug-treated group compared with the control group that revealed the cancer suppression mechanisms. Conclusions SSZ could improve CCA therapy by sensitization to CIS through killing CD44v9-positive cells and modifying the metabolic pathways, in particular tryptophan degradation (i.e., kynurenine pathway, serotonin pathway) and nucleic acid metabolism.

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.

Xanthohumol (XH), a plant flavonoid, was shown to attenuate cholangiocarcinoma (CCA) development induced by the liver fluke Opisthorchis viverrini (Ov) and N-dinitrosomethylamine (NDMA) in the hamster model. We investigated the possible involvement of autophagy, a self-degrading process dysregulated in cancer, in XH chemotherapeutic effect. During cholangiocarcinogenesis, the expression of LC3 (an autophagic marker) was increased in the precancerous stage and decreased in the cancerous stage. The XH-treated ON (Ov plus NDMA) group showed retarded progression of CCA along with increased expression of LC3. The possible relation between autophagy and cell death was investigated in cultured human CCA cells. XH induced apoptosis associated with reduced expression of BCL-2 and increased expression of BAX. In parallel, XH induced the autophagy flux, as testified by increased LC3-II and decreased p62, along with induction of BECLIN1 and Vps34. Inhibition of BECLIN1-dependent autophagy greatly limited XH toxicity in CCA cells. These data suggest that XH attenuates the development of CCA through overstimulation of autophagy which then precipitates apoptosis.

Abstract Background Cancer recurrence is the important problem of cholangiocarcinoma (CCA) patients, lead to a very high mortality rate. Therefore, the identification of candidate markers to predict CCA recurrence is needed in order to effectively manage the disease. This study aims to examine the predictive value of cancer stem cell (CSC) markers on the progression and recurrence of CCA patients. Methods The expression of 6 putative CSC markers, cluster of differentiation 44 (CD44), CD44 variant 6 (CD44v6), CD44 variants 8-10 (CD44v8-10), cluster of differentiation 133 (CD133), epithelial cell adhesion molecule (EpCAM), and aldehyde dehydrogenase 1A1 (ALDH1A1), was investigated in 178 CCA tissue samples using immunohistochemistry (IHC) and analyzed with respect to clinicopathological data and patient outcome including recurrence-free survival (RFS) and overall survival (OS). The candidate CSC markers were also investigated in serum from CCA patients, and explored for their predictive ability on CCA recurrence. Results Elevated protein level of CD44 and positive expression of CD44v6 and CD44v8-10 were significantly associated with short RFS and OS, while high levels of ALDH1A1 were correlated with a favorable prognosis patient. The elevated CD44v6 level was also correlated with higher tumor staging, whereas a decreasing level of ALDH1A1 was correlated with lower tumor staging. The levels of CD44, CD44v6 and CD44v8-10 were also correlated and were associated with a poor outcome. Furthermore, soluble CD44, CD44v6, CD44v8-10 and EpCAM were significantly increased in the recurrence group for early stage CCA; they also correlated with high levels of the tumor marker CA19-9. Elevated levels of CD44, CD44v6, CD44v8-10 or EpCAM alone or in combination has the potential to predict CCA recurrence. Conclusions The overexpression of CD44, CD44v6, CD44v8-10 and EpCAM increases predictability of post-operative CCA recurrence. Moreover, the overexpression of the panel of CSC markers combined with CA19-9 could improve our predictive ability for tumor recurrence in early stage CCA patients. This result may be beneficial for the patients in order to predict the outcome after treatment and may be useful for clinical intervention in order to improve patient survival.

Abstract Background Cancer recurrence is one of the most concerning clinical problems of cholangiocarcinoma (CCA) patients after treatment. However, an identification of predictive factor on Opisthorchis viverrini (OV)-associated CCA recurrence is not well elucidated. In the present study, we aimed to investigate the correlation of twelve targeted protein kinases with CCA recurrence. Methods Twelve protein kinases, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, 3, 4 (HER2, HER3, HER4), vascular endothelial growth factor receptor 3 (VEGFR3), vascular endothelial growth factor-C (VEGF-C), erythropoietin-producing hepatocellular carcinoma receptor type-A3 (EphA3), EphrinA1, phosphor-serine/threonine kinase 1 (p-Akt1), serine/threonine kinase 1 (Akt1), beta-catenin and protein Wnt5a (Wnt5a) were examined using immunohistochemistry. Pre-operative serum tumor markers, CA19–9 and CEA were also investigated. Results Among twelve protein kinases, EGFR, HER4, and EphA3 were associated with tumor recurrence status, recurrence-free survival (RFS) and overall survival (OS). Multivariate cox regression demonstrated that EGFR, HER4, EphA3 or the panel of high expression of these proteins was an independent prognostic factor for tumor recurrence. The combination of high expression of these proteins with a high level of CA19–9 could improve the predictive ability on tumor recurrence. Moreover, the patients were stratified more accurately when analyzed using the combination of high expression of these proteins with primary tumor (T) or lymph node metastasis (N) status. Conclusion EGFR, HER4, EphA3 or the panel of high expression of these proteins is an independent prognostic factor for post-operative CCA recurrence.

An aberrant regulation of lipid metabolism is involved in the pathogenesis and progression of cancer. Up-regulation of lipid biosynthesis enzymes, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and HMG-CoA reductase (HMGCR), has been reported in many cancers. Therefore, elucidating lipid metabolism changes in cancer is essential for the development of novel therapeutic targets for various human cancers. The current study aimed to identify the abnormal expression of lipid-metabolizing enzymes in cholangiocarcinoma (CCA) and to evaluate whether they can be used as the targets for CCA treatment. Our study demonstrated that a high expression of FASN was significantly correlated with the advanced stage in CCA patients. In addition, survival analysis showed that high expression of FASN and HMGCR was correlated with shorter survival of CCA patients. Furthermore, FASN knockdown inhibited the growth, migration and invasion in CCA cell lines, KKU055 and KKU213, as well as induced cell cycle arrest and apoptosis in the CCA cell lines. In addition, metabolomics study further revealed that purine metabolism was the most relevant pathway involved in FASN knockdown. Adenosine diphosphate (ADP), glutamine and guanine levels significantly increased in KKU213 cells while guanine and xanthine levels remarkably increased in KKU055 cells showing a marked difference between the control and FASN knockdown groups. These findings provide new insights into the mechanisms associated with FASN knockdown in CCA cell lines and suggest that targeting FASN may serve as a novel CCA therapeutic strategy.

CD44 is a transmembrane glycoprotein, the phosphorylation of which can directly trigger intracellular signaling, particularly Akt protein, for supporting cell growth, motility and invasion. This study examined the role of CD44 on the progression of Cholangiocarcinoma (CCA) using metabolic profiling to investigate the molecular mechanisms involved in the Akt signaling pathway. Our results show that the silencing of CD44 decreases Akt and mTOR phosphorylation resulting in p21 and Bax accumulation and Bcl-2 suppression that reduces cell proliferation. Moreover, an inhibition of cell migration and invasion regulated by CD44. Similarly, the silencing of CD44 showed an alteration in the epithelial-mesenchymal transition (EMT), e.g. an upregulation of E-cadherin and a downregulation of vimentin, and the reduction of the matrix metalloproteinase (MMP)-9 signal. Interestingly, a depletion of CD44 leads to metabolic pathway changes resulting in redox status modification and Trolox (anti-oxidant) led to the recovery of the cancer cell functions. Based on our findings, the regulation of CCA progression and metastasis via the redox status-related Akt signaling pathway depends on the alteration of metabolic profiling synchronized by CD44.

Background. Major hepatectomy is the mainstay of the treatment for cholangiocarcinoma. Infrahepatic inferior vena cava (IVC) clamping is an effective maneuver for reducing blood loss during liver transection. The impact of this procedure on major hepatectomy for cholangiocarcinoma is unknown. This study evaluated the effect of infrahepatic IVC clamping on blood loss during liver transection. Methods. Clinical and pathological data were collected retrospectively for 116 cholangiocarcinoma patients who underwent major hepatectomy between January 2015 and December 2016, to investigate the benefit of infrahepatic IVC clamping. Two of five surgeons adapted the policy performing infrahepatic IVC clamping during liver transection in all cases. Patients, therefore, were divided into those (n=39; 33.6%) who received infrahepatic IVC clamping during liver transection (C1) and those (n=77; 66.4%) who did not (C0). Results. The patients’ backgrounds, operative parameters, and extent of hepatectomy did not differ significantly between the 2 groups, except for gender. A significantly lower blood loss (p=0.028), blood transfusion (p=0.011), and rate of vascular inflow occlusion requirement (p

Summary:. In wound care, we usually focus nearly all of our efforts on the wound area while paying little attention to the periwound area. Although the periwound area may seem unimportant, it matters to patients. A female patient was admitted with a wound at the perianal area. Wound dressing was performed using standard wet-to-dry gauzes. The patient had several small complaints including irritant contact dermatitis, skin maceration, pain during dressing change, and fecal contamination to the wound. In this case, we ended up switching to a different method of wound dressing. We went from using wet-to-dry gauzes for the primary dressing to a hydrofiber with silver dressing and from gauze and Micropore as a secondary dressing to an adhesive sodium carboxymethylcellulose foam dressing. This resolved all complaints. The patient’s satisfaction score using visual analog scale increased from 2 to 10 (out of 10 points). This example shows how even small details can make a significant difference in wound care. Because periwound care is often neglected, therapeutic algorithm that integrates major challenges in periwound care into wound healing strategies is proposed.

Adjuvant chemotherapy is required for cholangiocarcinoma (CCA) patients after surgical treatment. Gemcitabine and gemcitabine plus cisplatin are considered the appropriate regimen; however, the response spectrum to chemotherapy differs between patients. Thus, the present study aims to evaluate the response pattern of individual CCA patients by using an in vitro method, histoculture drug response assay (HDRA), to predict the chemosensitivity of individual patients in a prospective study. Moreover, we also investigate the expression of gemcitabine and cisplatin sensitivity factors in CCA tissues in the same cases. Based on the dose response curve, 1000 and 1500 μg/ml of gemcitabine were used as the testing concentrations. For cisplatin, concentrations of 20 and 25 μg/ml were selected for testing and for the combination regimen, 1000 μg/ml of gemcitabine and 20 μg/ml of cisplatin were chosen. The median %IR of each drug was measured as the cut-off to categorize the response pattern into response and non-response groups. In addition, we compared the effectiveness of the chemotherapy regimens between gemcitabine alone and gemcitabine plus cisplatin. The %IR of the combination of gemcitabine and cisplatin was significantly higher than gemcitabine alone. The relationship between the expression level of gemcitabine and cisplatin sensitive factors and the individual response pattern as well as clinicopathological data of CCA patients were analyzed. The results indicated that a low expression of the gemcitabine sensitive factor hENT-1 was significantly associated with the non-response group in vitro (p = 0.002). Moreover, the low expression of hENT-1 was also significantly associated with advanced stages CCA in the patients (p = 0.025). A low expression of MT and ERCC1 was significantly correlated with the response group in the in vitro experiments (p = 0.015 and p = 0.037 for MT and ERCC1, respectively). Therefore, HDRA may serve as an aid to selecting chemotherapy, and the expression of hNET-1, MT and ERCC1 may serve as biomarkers for predicting chemotherapy success.

The analysis of cfDNA has been applied as a liquid biopsy in several malignancies. However, its value in the diagnosis and prognosis of cholangiocarcinoma (CCA) have not been well defined. We aimed to investigate the diagnostic and prognostic values of cfDNA level and tumor-specific mutation in circulating DNA (ctDNA) in CCA. The plasma cfDNA levels from 62 CCA patients, 33 benign biliary disease (BBD) patients and 30 normal controls were quantified by fluorescent assay. Targeted probe-based sequencing of 60 genes was applied for mutation profiling in 10 ctDNA samples and their corresponding treatment-naïve tissues. cfDNA levels in CCA were significantly higher than those in BBD and normal controls. We found that cfDNA levels at 0.2175 and 0.3388 ng/µL significantly discriminated CCA from healthy controls and BBD with 88.7 and 82.3% sensitivity and 96.7 and 57.6% specificity, respectively. cfDNA levels showed superior diagnostic efficacy in detecting CCA compared to CEA and CA19-9. ARID1A (30%), PBRM1 (30%), MTOR (30%), and FGFR3 (30%) mutations were the most common. Using nine frequently mutated genes in the ctDNA samples, the diagnostic accuracy of cfDNA sequencing was 90.8%, with 96.7% average sensitivity and 72.4% specificity. This study supports the use of cfDNA as a diagnosis and prognostic biomarker for CCA.

Potential biomarkers which include S100 calcium binding protein A9 (S100A9), mucin 5AC (MUC5AC), transforming growth factor β1 (TGF-β1), and angiopoietin-2 have previously been shown to be effective for cholangiocarcinoma (CCA) diagnosis. This study attempted to measure the sera levels of these biomarkers compared with carbohydrate antigen 19-9 (CA19-9). A total of 40 serum cases of CCA, gastrointestinal cancers (non-CCA), and healthy subjects were examined by using an enzyme-linked immunosorbent assay. The panel of biomarkers was evaluated for their accuracy in diagnosing CCA and subsequently used as inputs to construct the decision tree (DT) model as a basis for binary classification. The findings showed that serum levels of S100A9, MUC5AC, and TGF-β1 were dramatically enhanced in CCA patients. In addition, 95% sensitivity and 90% specificity for CCA differentiation from healthy cases, and 70% sensitivity and 83% specificity for CCA versus non-CCA cases was obtained by a panel incorporating all five candidate biomarkers. In CCA patients with low CA19-9 levels, S100A9 might well be a complementary marker for improved diagnostic accuracy. The high levels of TGF-β1 and angiopoietin-2 were both associated with severe tumor stages and metastasis, indicating that they could be used as a reliable prognostic biomarkers panel for CCA patients. Furthermore, the outcome of the CCA burden from the Classification and Regression Tree (CART) algorithm using serial CA19-9 and S100A9 showed high diagnostic efficiency. In conclusion, results have shown the efficacy of CCA diagnosis and prognosis of the novel CCA-biomarkers panel examined herein, which may prove be useful in clinical settings.

Similar to bile duct cancer or cholangiocarcinoma (CCA) in the western world, opisthorchiasis-associated CCA in Southeast Asia is an aggressive cancer with high mortality rates. It is known to cause a significant health burden in the opisthorchiasis region in Thailand and possibly throughout mainland Southeast. To reduce this health burden, a comprehensive prevention and control program for opisthorchiasis, as well as CCA, is required. In this review, our aim is to provide a brief update of the current situation regarding the natural history of opisthorchiasis and health burden of CCA in Southeast Asia. A comprehensive approach to tackling these issues being implemented in Thailand under the “Cholangiocarcinoma Screening and Care Program” is described. This comprehensive program consists of a three stage prevention and patient care program. The primary prevention component involves opisthorchiasis screening using a new and sensitive urine assay. The secondary prevention component involves screening for CCA and periductal fibrosis, with suspected CCA patients following the protocol for confirmation and appropriate treatment. Due to the eco-epidemiology of opisthorchiasis-induced CCA, the anticipated impacts and outcomes of the program include short-, medium-, and the long-term goals for the reduction of CCA incidence. To achieve long-term sustainable impacts, concerted efforts to raise social awareness and participating action by general public, non-government organizations, and government agencies are necessary. The strategic plans developed for this program can be expanded for use in other endemic areas as well as being a model for use in other chronic diseases.

Labile iron pool is a cellular source of ions available for Fenton reactions resulting in oxidative stress. Living organisms avoid an excess of free irons by a tight control of iron homeostasis. We investigated the altered expression of iron regulatory proteins and iron discrimination in the development of liver fluke–associated cholangiocarcinoma. Additionally, the levels of labile iron pool and the functions of transferrin receptor-1 on cholangiocarcinoma development were also identified. Iron deposition was determined using the Prussian blue staining method in human cholangiocarcinoma tissues. We investigated the alteration of iron regulatory proteins including transferrin, transferrin receptor-1, ferritin, ferroportin, hepcidin, and divalent metal transporter-1 in cholangiocarcinoma tissues using immunohistochemistry. The clinicopathological data of cholangiocarcinoma patients and the expressions of proteins were analyzed. Moreover, the level of intracellular labile iron pool in cholangiocarcinoma cell lines was identified by the RhoNox-1 staining method. We further demonstrated transferrin receptor-1 functions on cell proliferation and migration upon small interfering RNA for human transferrin receptor 1 transfection. Results show that Iron was strongly stained in tumor tissues, whereas negative staining was observed in normal bile ducts of healthy donors. Interestingly, high iron accumulation was significantly correlated with poor prognosis of cholangiocarcinoma patients. The expressions of iron regulatory proteins in human cholangiocarcinoma tissues and normal liver from cadaveric donors revealed that transferrin receptor-1 expression was increased in the cancer cells of cholangiocarcinoma tissues when compared with the adjacent normal bile ducts and was significantly correlated with cholangiocarcinoma metastasis. Labile iron pool level and transferrin receptor-1 expression were significantly increased in KKU-214 and KKU-213 when compared with cholangiocyte cells (MMNK1). Additionally, the suppression of transferrin receptor-1 expression significantly decreased intracellular labile iron pool, cholangiocarcinoma migration, and cell proliferation when compared with control media and control small interfering RNA. In Conclusion, high expression of transferrin receptor-1 resulting in iron uptake contributes to increase in the labile iron pool which plays roles in cholangiocarcinoma progression with aggressive clinical outcomes.

Objective: Endoscopic retrograde cholangiopancreatography (ERCP) is often used to provide internal biliary drainage in patients with unresectable perihilar cholangiocarcinoma; however, clinical success is not achieved in some patients. The aim of this study was to evaluate the factors associated with clinical success of ERCP with biliary drainage in unresectable perihilar cholangiocarcinoma. Methods: We retrospectively evaluated 68 patients with unresectable perihilar cholangiocarcinoma who underwent ERCP with biliary drainage from 2010 to 2018. Patients received either unilateral or bilateral metallic stents. Unsuccessful biliary drainage was defined as a reduction in serum bilirubin of less than 50% from baseline levels within 2 weeks. Student’s t-test and Pearson’s Chi-square test were utilized for analyses. Results: The rate of successful biliary drainage was 60.3%. Elective biliary drainage exhibited a significantly higher success rate compared to emergency biliary drainage [odds ratio (OR)=3.903, 95% CI, 1.212-12.566, P=.032]. High serum total bilirubin was associated with a significantly higher success rate compared to low serum total bilirubin (OR=1.069, 95% CI, 1.008-1.134, P=.025). Conclusion: Higher serum total bilirubin levels and elective intervention were identified as factors affecting the success rate of ERCP with biliary drainage. These factors should be assessed to identify patients who might benefit from this procedure. [ABSTRACT FROM AUTHOR]

Background: The liver fluke Opisthorchis viverrini (OV), which subsequently inhabits the biliary system and results in periductal fibrosis (PDF), is one of the primarily causes of cholangiocarcinoma (CCA), a bile duct cancer with an exceptionally high incidence in the northeast of Thailand and other Greater Mekong Subregion (GMS) countries. Insights in fecal metabolic changes associated with PDF and CCA are required for further molecular research related to gut health and potential diagnostic biological marker development. Methods: In this study, nuclear magnetic resonance (NMR) metabolomics was applied for fecal metabolic phenotyping from 55 fecal water samples across different study groups including normal bile duct, PDF and CCA groups. Results: By using NMR spectroscopy-based metabolomics, fecal metabolic profiles of patients with CCA or PDF and of individuals with normal bile duct have been established with a total of 40 identified metabolites. Further multivariate statistical analysis and hierarchical clustering heat map have demonstrated the PDF- and CCA-specific metabotypes through various altered metabolite groups including amino acids, alcohols, amines, anaerobic glycolytic metabolites, fatty acids, microbial metabolites, sugar, TCA cycle intermediates, tryptophan catabolism substrates, and pyrimidine metabolites. Compared to the normal bile duct group, PDF individuals showed the significantly elevated relative concentrations of fecal ethanol, glycine, tyrosine, and N-acetylglucosamine whereas CCA patients exhibited the remarkable fecal metabolic changes that can be evident through the increased relative concentrations of fecal uracil, succinate, and 5-aminopentanoate. The prominent fecal metabolic alterations between CCA and PDF were displayed by the reduction of relative concentration of methanol observed in CCA. The metabolic alterations associated with PDF and CCA progression have been proposed with the involvement of various metabolic pathways including TCA cycle, ethanol biogenesis, hexamine pathway, methanol biogenesis, pyrimidine metabolism, and lysine metabolism. Among them, ethanol, methanol, and lysine metabolism strongly reflect the association of gut-microbial host metabolic crosstalk in PDF and/or CCA patients. Conclusion: The PDF- and CCA-associated metabotypes have been investigated displaying their distinct fecal metabolic patterns compared to that of normal bile duct group. Our study also demonstrated that the perturbation in co-metabolism of host and gut bacteria has been involved from the early step since OV infection to CCA tumorigenesis. [ABSTRACT FROM AUTHOR]