Identification of novel biomarkers to distinguish clear cell and non-clear cell renal cell carcinoma using bioinformatics and machine learning.

Renal cell carcinoma (RCC), accounting for 90% of all kidney cancer, is categorized into clear cell RCC (ccRCC) and non-clear cell RCC (non-ccRCC) for treatment based on the current NCCN Guidelines. Thus, the classification will be associated with therapeutic implications. This study aims to identify novel biomarkers to differentiate ccRCC from non-ccRCC using bioinformatics and machine learning. The gene expression profiles of ccRCC and non-ccRCC subtypes (including papillary RCC (pRCC) and chromophobe RCC (chRCC)), were obtained from TCGA. Differential expression genes (DEGs) were identified, and specific DEGs for ccRCC and non-ccRCC were explored using a Venn diagram. Gene Ontology and pathway enrichment analysis were performed using DAVID. The top ten expressed genes in ccRCC were then selected for machine learning analysis. Feature selection was operated to identify a minimum highly effective gene set for constructing a predictive model. The expression of best-performing gene set was validated on tissue samples from RCC patients using immunohistochemistry techniques. Subsequently, machine learning models for diagnosing RCC were developed using H-scores. There were 910, 415, and 835 genes significantly specific for DEGs in ccRCC, pRCC, and chRCC, respectively. Specific DEGs in ccRCC enriched in PD-1 signaling, immune system, and cytokine signaling in the immune system, whereas TCA cycle and respiratory, signaling by insulin receptor, and metabolism were enriched in chRCC. Feature selection based on Decision Tree Classifier revealed that the model with two genes, including NDUFA4L2 and DAT, had an accuracy of 98.89%. Supervised classification models based on H-score of NDUFA4L2, and DAT revealed that Decision Tree models showed the best performance with 82% accuracy and 0.9 AUC. NDUFA4L2 expression was associated with lymphovascular invasion, pathologic stage and pT stage in ccRCC. Using integrated bioinformatics and machine learning analysis, NDUFA4L2 and DAT were identified as novel biomarkers to differential diagnosis ccRCC from non-ccRCC.

Anticancer properties and metabolomic profiling of Shorea roxburghii extracts toward gastrointestinal cancer cell lines.

BACKGROUND: Gastrointestinal cancer (GIC) ranks as the highest cause of cancer-related deaths globally. GIC patients are often diagnosed at advanced stages, limiting effective treatment options. Chemotherapy, the common GIC recommendation, has significant disadvantages such as toxicity and adverse effects. Natural products contain substances with diverse pharmacological characteristics that promise for use in cancer therapeutics. In this study, the flower of renowned Asian medicinal plant, Shorea roxburghii was collected and extracted to investigate its phytochemical contents, antioxidant, and anticancer properties on GIC cells. METHODS: The phytochemical contents of Shorea roxburghii extract were assessed using suitable methods. Phenolic content was determined through the Folin-Ciocalteu method, while flavonoids were quantified using the aluminum chloride (AlCl3) method. Antioxidant activity was evaluated using the FRAP and DPPH assays. Cytotoxicity was assessed in GIC cell lines via the MTT assay. Additionally, intracellular ROS levels and apoptosis were examined through flow cytometry techniques. The correlation between GIC cell viability and phytochemicals, 1H-NMR analysis was conducted. RESULTS: Among the four different solvent extracts, ethyl acetate extract had the highest phenolic and flavonoid contents. Water extract exhibited the strongest reducing power and DPPH scavenging activity following by ethyl acetate. Interestingly, ethyl acetate extract demonstrated the highest inhibitory activity against three GIC cell lines (KKU-213B, HepG2, AGS) with IC50 values of 91.60 µg/ml, 39.38 µg/ml, and 35.59 µg/ml, while showing less toxicity to normal fibroblast cells. Ethyl acetate extract induced reactive oxygen species and apoptosis in GIC cell lines by downregulating anti-apoptotic protein Bcl-2. Metabolic profiling-based screening revealed a positive association between reduced GIC cell viability and phytochemicals like cinnamic acid and its derivatives, ferulic acid and coumaric acid. CONCLUSIONS: This study highlights the potential of natural compounds in Shorea roxburghii in the development of more effective and safer anticancer agents as options for GIC as well as shedding light on new avenues for cancer treatment.

Pathology of Cholangiocarcinoma.

The liver excretes bile through the biliary system, which has a complicated anatomical structure. Cholangiocarcinoma, a malignant bile duct epithelial tumor, is separated into intrahepatic and extrahepatic portions depending on the structure of the bile duct and exhibits both similarities and varieties in patient presentations and staging. The three main macroscopic characteristics of cholangiocarcinoma-mass formating, intraductal growth, and periductal infiltrating types-allow pathologists and surgeons to see and analyze the cancerous tissue. The majority of cholangiocarcinoma patients are in advanced stages and poor prognosis. Although surgery is the main treatment option, target therapy based on molecular pathology background offers hope for improving patient's prognosis.

Alteration of STK11 Expression Associated With Cholangiocarcinoma Progression.

BACKGROUND/AIM: Serine/threonine kinase 11 (STK11), a tumor suppressor, controls 5' AMP-activated protein kinase (AMPK) signaling in a variety of cellular functions. Mutated STK11 has been identified as a novel driver gene that promotes cancer progression. The purpose of this study was to investigate the alteration of STK11 and its correlation with clinicopathological data in cholangiocarcinoma (CCA). MATERIALS AND METHODS: Gene mutation and expression analyses were performed using cBioportal and Gene Expression Profiling Interactive Analysis version 2 (GEPIA2). qRT-PCR was performed to measure STK11 mRNA levels and immunohistochemistry was performed to investigate STK11 protein expression in CCA tissues. RESULTS: The results from publicly available cancer datasets showed that 2.7% of CCA cases had STK11 mutations. Most of STK11 gene mutations are of the truncating type and result in low STK11 mRNA and protein expression. We detected a correlation between STK11 mutation status and the tendency for shorter patient survival. The results of qRT-PCR revealed that STK11 mRNA levels were statistically significantly lower in CCA patients with mutated STK11 compared to those with wild-type STK11 (p-value=0.013). Immunohistochemical staining showed high STK11 expression in 43.8% and low expression in 56.2% of CCA tissues examined. Low STK11 protein expression resulted in poor prognosis compared with high STK11 expression, especially in CCA papillary carcinoma. Univariate and multivariate analysis revealed that high STK11 expression was associated with a decreased hazard ratio of patient survival rates (HR=0.696, p-value=0.06 and HR=0.666, p-value=0.04, respectively). CONCLUSION: Alteration of STK11 mutational or mRNA/protein status might be used as a potential predictive biomarker for the prognosis of the clinical outcomes in CCA patients.

Diagnosis of Metastatic Carcinoma Using Body Cavity Fluid Specimens: A Comparison of Diagnostic Panels.

INTRODUCTION: Body cavity effusions are routinely used as cytologic specimens. The distinction between metastatic carcinoma, mesothelioma, and reactive mesothelial cells remains a major challenge. Immunohistochemistry (IHC) is a supplemental method that can aid in diagnosis and often involves many markers as part of an IHC panel. Several immunohistochemical markers are now widely used. This study aims to determine the optimal immunomarkers and IHC panels to differentiate reactive mesothelial cells from metastatic cancer in body cavity fluid samples. METHODS: IHC was performed for claudin-4, MOC-31, Ber-Ep4, D2-40, and calretinin on sections derived from 152 cellblocks containing effusions. The samples consisted of 16 (10.53%) benign and 136 (89.47%) malignant tumors, including 87 (63.97%) lung cancers, nine (6.62%) breast cancers, 11 (8.09%) gynecologic cancers, seven (5.15%) pancreaticobiliary cancers, and 22 (16.17%) unspecified primary malignancies. RESULTS: Claudin-4, MOC-31, Ber-EP4, D2-40, and calretinin demonstrated sensitivities of 91.18%, 91.91%, 55.88%, 90.44%, and 98.53%, respectively. The corresponding specificities were 100.00%, 100.00%, 100.00%, 93.75%, and 100.00%. The sensitivity and specificity were both 100% when claudin-4 or MOC-31 was combined with calretinin. The combination of four markers as an IHC panel (claudin-4, MOC-31, calretinin, and D2-40) had a sensitivity of 97.79% and a specificity of 100.00%. CONCLUSION: Claudin-4 and MOC-31 both demonstrated significant diagnostic value in distinguishing metastatic epithelial carcinoma from reactive mesothelium. The sensitivity, specificity, and accuracy of these two markers, one of which is an epithelial marker and one of which is a mesothelial marker, reached 100%. Therefore, a combination of these two markers may be appropriate.

Prognostic significance of tumor-infiltrating lymphocytes in predicting outcome of distal cholangiocarcinoma in Thailand.

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.

Modification of the eighth AJCC/UICC staging system for perihilar cholangiocarcinoma: An alternative pathological staging system from cholangiocarcinoma-prevalent Northeast Thailand.

Aim: This 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. Method: Four 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. Results: Multivariate 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.). Conclusion: The 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.

Modification of the AJCC/UICC 8th edition staging system for intrahepatic cholangiocarcinoma: proposal for an alternative staging system from cholangiocarcinoma-prevalent Northeast Thailand.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) arises from bile ducts within the liver. Thailand has the highest incidence of CCA worldwide, with a high mortality rate. Early diagnosis and accurate prognostic stratification can improve overall survival. We aim to modify the AJCC/UICC 8th edition staging system for iCCA by creating the Khon Kaen University (KKU) staging system for more precise patient stratification and prognostic prediction. METHODS: A total of 298 iCCA patients who underwent hepatectomy were included in this retrospective study at the Srinagarind Hospital, Khon Kaen University, Thailand. Univariate and multivariate analysis were performed to examine survival rate, hazard ratio, and prognostic factors. RESULTS: Univariate and multivariate analysis of the cohort showed that growth patterns, histological type, histological grade, lymph node metastasis and distant metastasis were independent prognostic factors when compared to the respective reference groups. The 8th AJCC staging system incorporated growth patterns into the KKU staging system. This model modified AJCC stages I, II, and III for better prediction of patient survival. CONCLUSION: Growth patterns were incorporated to improve the 8th AJCC staging system for prognostication of iCCA patients in Northeast Thailand. We propose the KKU staging system as an alternative model for iCCA staging to augment the accuracy of survival prognostication.

Prognostic Significance of Growth Pattern in Predicting Outcome of Opisthorchis viverrini-Associated Distal Cholangiocarcinoma in Thailand.

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.

The Impact of Pre-analytical Quality Initiatives on Cholangiocarcinoma Diagnostics in Thailand.

Cholangiocarcinoma (CCA) is the most prevalent malignancy in Thailand, with unfortunate late diagnosis and frequent metastatic disease outcomes. An accurate tissue diagnosis is the first and most important step in the treatment of CCA. Tissue quality and preservation during the pre-analytical phase play major roles in the proper histological evaluation and potential biomarker testing. This study evaluated the impact of using the "Cholangiocarcinoma Screening and Care Program (CASCAP)" container, as an innovative tool to address pre-analytical challenges faced by pathology laboratories in Thailand. This is a comparison study examining the quality of CCA specimens using the CASCAP container vs. the conventional method, using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). CCA tissue quality using the CASCAP container significantly reduced artifact deposition while improving the cellular structure and nuclear and cytoplasmic morphologies. The immunohistochemical expression of cytokeratin 19 (CK19), a prognostic marker in CCA, significantly improved in the CASCAP container group in comparison with the conventional method. This innovation is proven to significantly enhance the CCA tissue quality diagnostics and prognostic biomarker testing, hence improving overall cancer care, diagnosis, and treatment in Thailand.

Chromosomal aberrations, visualized using UroVysion® fluorescence in-situ hybridization assay, can predict poor prognosis in formalin-fixed paraffin-embedded tissues of cholangiocarcinoma patients.

Cholangiocarcinoma (CCA) is a highly aggressive malignant tumor that has highest incidence in northeastern Thailand. The survival rate of CCA patients after receiving surgical treatment is quite low. Recently, genetic alterations including chromosome abnormalities have been studied as predictive factors and to aid planning for further treatment. This study aims to investigate the association between chromosomal aberrations, clinical data, and overall survival time of CCA patients. Formalin-fixed paraffin-embedded (FFPE) tissues from 194 CCA patients were examined. The copy numbers of chromosomes 3, 7, 17 and 9p21 were investigated using the UroVysion® fluorescence in-situ hybridization (FISH) assay. The overall survival time (OS) of CCA patients with or without polysomy of chromosomes 3, 7, 17 and/or loss of 9p21 were statistically analyzed in association with their clinicopathological parameters. Kaplan-Meier analysis was performed. The OS of patients with polysomy of chromosomes 3 + 7 was significantly shorter than those without this polysomy (log-rank P = 0.006; median OS 14.79 vs. 19.62 months). Moreover, patients with polysomy of chromosomes 3 + 7+17 and heterozygous for 9p21 loss have significantly shorter survival time than those without such chromosomal aberrations (log-rank P = 0.001; median OS 15.74 vs. 37.57 months). Interestingly, multivariate analysis revealed that polysomy of chromosomes 3 + 7 and of chromosomes 3 + 7+17 with 9p21 heterozygous loss were independent predictive factors of a poor OS (P = 0.027; P = 0.008, respectively).The chromosomal aberrations patterns which we evaluated using FISH; 1) polysomy of chromosomes 3 + 7 and 2) polysomy of chromosomes 3 + 7+17 with 9p21 heterozygous loss, have strong potential as indicators of poor prognosis in CCA patients.

Fluorescence in situ hybridization detection of chromosome 7 and/or 17 polysomy as a prognostic marker for cholangiocarcinoma.

Cholangiocarcinoma (CCA) is highly endemic in the Northeast Thailand. Recently, chromosome aberrations provided new insights into pathogenesis of CCA. Therefore, chromosome aberration might be used as a prognostic factor and therapeutic planning of this cancer. This aim of this study is to examine the correlation between an increase of chromosome 7 (C7) and/or 17 (C17) copy number variants (CNVs) with clinicopathological data and the overall survival time (OS) of CCA patients using fluorescence in situ hybridization (FISH) assays. C7 and C17 CNVs were examined using FISH form 157 formalin-fixed paraffin-embedded (FFPE) tissues of CCA patients from Khon Kaen, Thailand between 2011 and 2015. OS was visualized using Kaplan-Meier plot. Univariate and multivariate analyses were used to determine the ability of the clinicopathological parameters to predict OS. C17 > trisomy (odd ratio, 6.944, P < 0.001), C7/17 trisomy (odd ratio; 4.488, P = 0.019), and C7/17 > trisomy (odd ratio; 6.723, P < 0.001) were independently predictive factors for lymph node metastasis. Interestingly, an increase of C7, C17, and C7/17 CNVs in both trisomy and > trisomy was independently correlated with short median OS. An increased of C7 and/or 17 have a potential as a poor prognostic marker in CCA patients.

High Levels of Serum IgG for Opisthorchis viverrini and CD44 Expression Predict Worse Prognosis for Cholangiocarcinoma Patients after Curative Resection.

BACKGROUND: Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA) has a high immune response with chronic inflammation and oxidative stress. CD44 and Nestin, two cancer stem cell (CSC) markers, play major roles in cancer cell survival. Effects of immune response and expression CSC markers on survival of patients with CCA remain unclear. OBJECTIVE: To investigate the effects of level of OV IgG together with CSC marker expression and also the combination of these markers on survival of CCA patients after curative resection. METHODS: All serum specimens from CCA patients who underwent curative surgery from 2005 to 2015 were examined for IgG for OV antigen by ELISA. Tissue specimens were studied for CD44 and Nestin expression. Survival analysis by Cox proportional hazard model was used for estimating hazard ratio (HR) with a 95% confidence interval (CI). RESULTS: In this study, 122 (69.3%) of 176 were positive for OV IgG, and 35 (19.9%) were considered to have high-positive OV IgG. CD44s positive expression was found in 54 (40%), CD44v6 high expression in 96 (69.6%), CD44v8-10 high expression in 87 (63.5%) and Nestin high expression in 21 (16.1%). Multivariate survival analysis found that high-positive OV IgG and late stage tumor were independent prognostic factors with the adjusted HR of 2.24 (95% CI 1.27-3.93) and 2.78 (95% CI 1.46-5.29), respectively. Subgroup analysis in early and late stage CCA showed that a combined positive OV IgG and CD44s expression with the high expression of CD44v8-10 had the significantly poorest prognosis with HR of 3.75 (95% CI 1.61-8.72) and HR of 1.76 (95% CI 1.02-3.03), respectively. CONCLUSION: A high level of OV IgG as well as a high level of CSC markers resulted in an aggressive CCA. OV IgG level together with CSC markers can be used as the prognostic markers for CCA patients' survival. The study of the CD44 pathway is promising for adjuvant treatment.

CD44 modulates metabolic pathways and altered ROS-mediated Akt signal promoting cholangiocarcinoma progression.

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.

Sulfasalazine modifies metabolic profiles and enhances cisplatin chemosensitivity on cholangiocarcinoma cells in in vitro and in vivo models.

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.

Metabolic Profiling of Praziquantel-mediated Prevention of Opisthorchis viverrini-induced Cholangiocyte Transformation in the Hamster Model of Cholangiocarcinoma.

BACKGROUND: Opisthorchis viverrini (Ov) infection-induced cholangiocarcinoma (CCA) is a major public health problem in northeastern Thailand. Praziquantel was shown to prevent CCA development in an Ov-infected hamster model; however, the molecular mechanism remains unknown. MATERIALS AND METHODS: In this study, we used a hamster model with Ov and N-nitrosodimethylamine-induced CCA to study the mechanisms of praziquantel action. The liver tissues from the hamsters with and without praziquantel treatment were analyzed using 1H nuclear magnetic resonance spectroscopy. RESULTS: A total of 14 metabolites were found to be significantly different between the two groups. Furthermore, the combination of acetate, inosine and sarcosine was shown to exert an anti-inflammatory effect through interleukin-6 inhibition in a macrophage cell line, suggesting a mechanism by which praziquantel may prevent inflammation caused by Ov, cholangiocyte transformation and further CCA develpoment. CONCLUSION: These findings might avail the development of a preventive strategy for CCA in high-risk populations.

Curative effect of xanthohumol supplementation during liver fluke-associated cholangiocarcinogenesis: Potential involvement of autophagy.

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.

In vitro and in vivo Anti-Tumor Effects of Pan-HER Inhibitor Varlitinib on Cholangiocarcinoma Cell Lines.

BACKGROUND: Cholangiocarcinoma (CCA) is a slowly progressing but highly aggressive malignancy. Targeting the HER protein family represents a potential therapeutic strategy for CCA treatment. The pan-HER inhibitor varlitinib is being developed for the treatment of breast cancer, gastric cancer, and biliary tract cancer, which includes CCA. This study aims to evaluate the anti-tumor effect of varlitinib on CCA using both in vitro and in vivo models. MATERIALS AND METHODS: HER family expression profiles and the cytotoxic activity of varlitinib were determined in CCA cell lines (KKU-214, KKU-213, KKU-156 and KKU-100) and cholangiocyte (MMNK-1). Anti-proliferation and apoptosis induction were examined in KKU-214 and KKU-100 cell lines. A combination of varlitinib with PI3K inhibitor, BKM-120, was explored for efficacy in the KKU-100 cell line. In addition, the anti-tumor activity of varlitinib on CCA and the key metabolites were evaluated in tumor tissues from CCA xenograft model. RESULTS: Elevated expressions of EGFR and HER2 were observed in KKU-214 and KKU-100 cells and varlitinib can suppress CCA cell growth in the micromolar range. Varlitinib inhibits cell proliferation and enhances cell death via the suppression of Akt and Erk1/2 activity in the KKU-214 cell line. While KKU-100 cells showed a poor response to varlitinib, a combination of varlitinib with BKM-120 improved anti-tumor activity. Varlitinib can significantly suppress tumor growth in the CCA xenograft model after oral administration for 15 days without noticeable toxicity, and aspartate can be the key metabolite to correlate with varlitinib response. CONCLUSION: Our study indicates that varlitinib is a promising therapeutic agent for CCA treatment via the inhibition of EGFR/HER2. The anti-tumor effect of varlitinib on CCA also showed synergism in combination with PI3K inhibition. Aspartate metabolite level was correlated with varlitinib response. Combination of varlitinib with targeted drug or cytotoxic drug was recommended.

A panel of protein kinase high expression is associated with postoperative recurrence in cholangiocarcinoma.

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.

Overexpression of a panel of cancer stem cell markers enhances the predictive capability of the progression and recurrence in the early stage cholangiocarcinoma.

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.