Extrahepatic Cholangiocarcinoma Tissues Research Articles

Unraveling the heterogeneity of intercellular crosstalk in intrahepatic and extrahepatic cholangiocarcinoma using single-cell and spatial transcriptome profiling.

50 Background: Extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC) are two types of malignant tumors that arise from the bile duct epithelium at different locations. Currently, effective treatment options for these tumors are limited, and their microenvironmental differences remain poorly understood. Methods: We collected retrospective clinicopathological data of ECC (N=64) and ICC (N=153) patients from our hospital and integrated public database information to analyze the clinical characteristics, survival, and prognosis of ECC and ICC. We performed single-cell sequencing (scRNA-seq) and spatial transcriptomic techniques on three ECC and three ICC tumor tissues. Verification of scRNA-seq results was done using immunohistochemistry, flow cytometry, and polychromatic immunofluorescence. Additionally, we analyzed RNA and ATAC sequencing data for exhausted CD8+ T cells TEX obtained from ICC and ECC tissue samples. We used InferCNV to infer copy number variations (CNVs) of epithelial cells from ICC and ECC. Results: Our results showed that ECC and ICC differ in their clinical characteristics such as age, gender, bilirubin, CEA, and outcome. ECC had higher copy number amplification variation and exhibited inflammatory signaling and ongoing immunoediting, reflected in higher immune checkpoint expression such as PDCD1, CTLA4, and LAG3, and tumor infiltration with highly differentiated dysfunctional PDCD1+ LAG3+ CXCL13+ exhausted CD8+ T cells (TEX) and precursors of exhausted T (TPEX). RNA velocity analysis revealed different developmental trajectories of T cells in ECC and ICC, predominantly in naive/stem-like and memory T cells. We also identified a trefoil factor 3 (TFF3)-positive epithelial subpopulation in ECC. TFF3+ epithelial cells had closer interaction with CTHRC1+ fibroblasts, which induced more CD8+ T cells to TEX and TPEX. TFF3+ cells exhibited higher CNV scores and higher PD-L1 and PD-L2 expression, and co-localized with TEX and TPEX, providing one explanation for the immunosuppressive environment in ECC and suggesting a potential response to immune checkpoint therapy (ICT). Conclusions: Our study provides a comprehensive characterization of the cellular heterogeneity landscape of ECC and ICC, which can guide the optimization of personalized cancer immunotherapy. We identified potential biomarkers and targets for immunotherapies, and our findings shed light on the underlying mechanisms of tumor immune evasion and provide a foundation for further research in this area.

Differential Expression of Long Non-coding RNA Cancer Susceptibility Candidate 2 and Imprinted Gene H19 in Extrahepatic Cholangiocarcinoma

Objective To investigate the expression and the potential roles of long non-coding RNA(lncRNA)cancer susceptibility candidate 2(CASC2)and imprinted gene H19 in extrahepatic cholangiocarcinoma(ECC). Methods Four samples from patients with ECC were collected for high-throughput sequencing which was conducted to reveal the transcriptomic profiles of lncRNA CASC2 and H19.Bioinformatics tools were employed to predict the potential roles of the two genes.Another 22 ECC tissue samples and the cholangiocarcinoma cell lines(RBE,QBC939,HuH-28,and HuCCT1)with different degrees of differentiation were selected for validation.The para-carcinoma tissue and normal human intrahepatic biliary epithelial cell(HIBEC)were used as the control groups.The expression levels of lncRNA CASC2 and H19 in carcinoma tissue,para-carcinoma tissue,and cell lines were determined by real-time quantitative polymerase chain reaction(qRT-PCR).The correlation analysis was carried out for the clinical indicators of patients with the expression levels of the target genes. Results The two target genes showed significantly different expression between carcinoma tissue and para-carcinoma tissue(all P<0.05).Specifically,CASC2 had higher expression level in the carcinoma tissue than in the para-carcinoma tissue(t=1.262,P=0.025),whereas the expression of H19 showed an opposite trend(t=1.285,P=0.005).The expression levels of CASC2 in QBC939(t=8.114,P=0.015)and HuH-28(t=9.202,P=0.012)cells were significantly higher than that in the control group.The expression levels of H19 were significantly lower in RBE(t=-10.244,P<0.001),QBC939(t=-10.476,P<0.001),HuH-28(t=-19.798,P<0.001),and HuCCT1(t=-16.193,P=0.004)cells than in the control group.Bioinformatics analysis showed that CASC2 was mainly involved in the metabolic process and H19 in the development of multicellular organisms.Both CASC2 and H19 were related to catalytic activity.The expression level of lncRNA CASC2 was correlated with pathological differentiation(χ 2=6.222,P=0.022)and lymph node metastasis(χ2=5.455,P=0.020),and that of lncRNA H19 with pathological differentiation(χ2=1.174,P=0.029)and tumor size(χ2=-0.507,P=0.037). Conclusions In the case of ECC,lncRNA CASC2 and H19 have transcription disorders.lncRNA CASC2 is generally up-regulated in the carcinoma tissue,while H19 is down-regulated.Both genes have the potential to become new molecular markers for ECC.

TGR5 promotes cholangiocarcinoma by interacting with mortalin

Takeda-G-protein-receptor-5 (TGR5) is a G-protein-coupled receptor (GPCR) activated by bile acids, and mortalin is a multipotent chaperone of the HSP70 family. In the present study, TGR5 was detected by immunohistochemistry (IHC) in extrahepatic cholangiocarcinoma (ECC) specimens, and TGR5 expression in ECC tissues and adjacent tissues was compared. In vitro TGR5 was overexpressed and knocked down in human intrahepatic cholangiocarcinoma (ICC) cell line RBE and human extrahepatic cholangiocarcinoma (ECC) cell line QBC-939 to observe its effects on the biological behavior of cholangiocarcinoma (CC) cells, including proliferation, apoptosis and migration. In vivo xenograft model was constructed to explore the role of TGR5 in CC growth. Proteins that interacted with TGR5 were screened using an immunoprecipitation spectrometry approach, and the identified protein was down-regulated to investigate its contribution to CC growth. The present study demonstrated that TGR5 is highly expressed in CC tissues, and strong TGR5 expression may indicate high malignancy in CC. Furthermore, TGR5 promotes CC cell proliferation, migration, and apoptosis resistance. TGR5 boosts CC growth in vivo. In addition, TGR5 combines with mortalin and regulates mortalin expression in the CC cell line. Mortalin participates in the TGR5-induced increase in CC cell proliferation. In conclusion, TGR5 is of clinical significance based on its implications for the degree of malignancy in patients with CC. Mortalin may be a downstream component regulated by TGR5, and TGR5 promotes cholangiocarcinoma at least partially by interacting with mortalin and upregulating its expression. Both TGR5 and mortalin are positive regulators, and may serve as potential therapeutic targets for CC.

BIRC7 and STC2 Expression Are Associated With Tumorigenesis and Poor Outcome in Extrahepatic Cholangiocarcinoma.

Background:Extrahepatic cholangiocarcinoma (EHCC) is a highly aggressive epithelial malignancy and has a poor prognosis for the insensitivity to therapies and difficulty in detection. Novel targets and biomarkers are urgently needed to develop for functional, diagnostic and prognostic application on EHCC.Methods:Immunohistochemical staining technique using the EnVision antibody complex was performed on the samples obtained from 100 EHCC, 30 peritumoral extrahepatic biliary tract (EHBT), 10 EHBT adenomas and 15 normal EHBT tissues.Results:The positive rates of BIRC7 and STC2 expression in tissues obtained from peritumoral EHBT, EHBT adenomas and normal EHBT were significantly lower than those in EHCC tissues. BIRC7 and STC2 proteins were expressed at significantly higher levels in patients with lymph node metastasis, invasion of adjacent tissues, and higher TNM stage (III and/or IV) and unable to undergo resection (biopsy only). Kaplan-Meier survival curves indicated that significantly decreased overall survival rate in patients with positive-BIRC7 or positive-STC2 expression compared with patients of negative-BIRC7 or negative-STC2 expression, respectively. Cox-proportional regression analysis demonstrated that positive-BIRC7 and positive-STC2 expression, along with poor differentiation of EHCC, tumor size >3 cm, lymph node metastasis, invasion of adjacent tissues and unable to undergo resection are independent prognostic factors of EHCC patients.Conclusions:The levels of BIRC7 and STC2 expression were correlated with clinicopathological characteristics of EHCC, and positive expression of BIRC7 and STC2 are associated with progression and poor clinical outcomes of EHCC. BIRC7 and STC2 might be a potential biomarker for EHCC in clinic.

Prognostic and Clinicopathological Significance of EphB3 and Dysadherin Expression in Extrahepatic Cholangiocarcinoma.

AimEphB3 and dysadherin are involved in tumorigenesis and progression of many neoplasms. However, the roles of EphB3 and dysadherin in extrahepatic cholangiocarcinoma (ECC) remain to be revealed. In this study, we aimed to evaluate the expression of EphB3 and dysadherin, and investigate their clinicopathological significance in ECC.MethodsWe examined EphB3 and dysadherin expression in 100 ECC, 30 peritumoral tissues, 10 adenoma and 15 normal biliary tract tissues using EnVision immunohistochemistry. The relationship between EphB3 or dysadherin expression and clinicopathological features was evaluated using the χ2 test or Fisher’s exact test. The overall survival of ECC patients was analyzed using Kaplan-Meier univariate survival analysis and Log rank tests.ResultsWe found that EphB3 expression was significantly down-regulated and dysadherin expression was significantly up-regulated in ECC tissues compared with normal tissues (P < 0.01). EphB3 expression was negatively correlated with dysadherin expression in ECC (P < 0.01). The positive rate of EphB3 expression and negative rate of dysadherin expression was significantly higher in patients with well-differentiated type, no lymph node metastasis, no surrounding tissues and organs invasion, early TNM stages (I + II) and radical resection (P < 0.01). The survival of ECC patients with positive EphB3 or negative dysadherin expression was significantly longer than patients with negative EphB3 or positive dysadherin expression (P < 0.01). Cox multivariate analysis demonstrated that negative EphB3 or positive dysadherin expression were independent poor prognostic factors in ECC patients. The ROC curves suggested that EphB3 and dysadherin combined diagnostic efficacy (AUC=0.688, 95%CI: 0.603-0.772) was significantly higher EphB3 diagnostic efficacy (AUC=0.654, 95%CI: 0.564-0.743) or dysadherin diagnostic efficacy (AUC=0.648, 95%CI: 0.558-0.737) alone.ConclusionEphB3 and dysadherin are involved in the carcinogenesis and progression of ECC, and ECC patients with negative EphB3 or positive dysadherin expression have a poor prognosis.

Long noncoding RNA PCAT1 regulates extrahepatic cholangiocarcinoma progression via the Wnt/β-catenin-signaling pathway

Extrahepatic cholangiocarcinoma (ECC) is a deadly disease that often responds poorly to conventional chemotherapy or radiotherapy. Long noncoding RNAs (lncRNAs) play important roles in human cancers, including ECC, and recent studies indicated that the lncRNA prostate cancer-associated transcript 1 (non-protein coding) (PCAT1) is involved in multiple cancers. However, the role of PCAT1 in ECC is unclear. Previously, we showed that PCAT1 is up-regulated in both ECC tissue samples and cell lines. Here, we showed that downregulation of PCAT1 following transfection with silencing RNA reduced ECC cell growth and increased cell apoptosis. Additionally, PCAT1 suppression inhibited ECC cell migration and invasion as determined by transwell assay. Furthermore, we determined that PCAT1 is a competing endogenous for microRNA (miR)-122, with bioinformatics analysis and luciferase-reporter assay results demonstrating that PCAT1 regulated WNT1 expression via miR-122. Moreover, PCAT1 downregulation increased levels of glycogen synthase kinase 3β and significantly decreased β-catenin levels in whole cell lysates and nuclear fractions, indicating that PCAT1 silencing inhibited the Wnt/β-catenin-signaling pathway. We also observed that exogenous expression of WNT1 reversed PCAT1-silencing-induced inhibition of ECC cell growth inhibition. These results indicated that PCAT1 silencing inhibited ECC progression by reducing Wnt/β-catenin signaling through miR-122 repression and WNT1 expression. Our findings revealed an important role of PCAT1 in ECC and suggested that PCAT1 might be a potential ECC-related therapeutic target.

PD-L1 and PD-1 expression correlate with prognosis in extrahepatic cholangiocarcinoma.

The present study aimed to investigate the clinicopathological significance of programmed cell death ligand-1 (PD-L1) and programmed cell death protein 1 (PD-1) expression in extrahepatic cholangiocarcinoma (ECC). PD-L1 and PD-1 expression was detected by immunohistochemical methods in 70 ECC formalin-fixed, paraffin-embedded tissue specimens and 50 para-carcinoma tissue specimens. The associations of PD-L1 and PD-1 expression with clinicopathological characteristics and prognosis of ECC patients were explored. Positive rates of PD-L1 and PD-1 expression were increased in ECC tissues compared with those in the corresponding para-carcinoma tissues. Besides, the expression of PD-L1 was correlated with the expression of PD-1 (P<0.05). Statistical analysis revealed that the expression of PD-L1 and PD-1 in ECC tissues exhibited no correlation with patient age, sex or histological grade, but was significantly correlated with tumor-node-metastasis (TNM) stage and lymphatic metastasis. Univariate analysis demonstrated that PD-L1 expression, PD-1 expression, TNM stage and lymphatic metastasis were significantly associated with the survival time of patients. Further multivariate analysis revealed the PD-L1 expression was an independent prognostic factor of patients with ECC. These preliminary results suggested that PD-L1 or PD-1 immunodetection may be a valuable prognostic marker for ECC patients, and that PD-L1 immunodetection may be used as an independent factor to evaluate the prognosis of ECC patients.

MicroRNA-34a inhibits epithelial mesenchymal transition in human cholangiocarcinoma by targeting Smad4 through transforming growth factor-beta/Smad pathway.

BackgroundExtrahepatic Cholangiocarcinoma (EHCC) is one of the uncommon malignancies in the digestive system which is characterized by a poor prognosis. Aberrations of miRNAs have been shown involved in the progression of this disease. In this study, we evaluated the expression and effects of miR-34a on EHCC.MethodsmiR-34a expression levels were detected in EHCC tissues, adjacent non-tumor tissues, normal bile duct (NBD) specimens of patients and cholangiocarcinoma (CC) cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Relationships between miR-34a with clinical characteristics of EHCC patients were further analyzed. Computational search, functional luciferase assay and western blot were further used to demonstrate the downstream target of miR-34a in CC cells. Immunohistochemistry was carried on to identify the downstream target gene of miR-34a in EHCC patients. Cell morphology, invasion and migration assays were further applied to confirm the anti-carcinogenic effects of miR-34a through the downstream target.ResultsmiR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues and normal bile duct tissues. miR-34a was found correlated with the migration and invasion in EHCC patients. Smad4 was over-expressed in most of the EHCC patients and was further demonstrated as one of the downstream targets of miR-34a, which was involved in the progression of EHCC. Moreover, activation of miR-34a suppressed invasion and migration through TGF-beta/Smad4 signaling pathway by epithelial-mesenchymal transition (EMT) in vitro.ConclusionsTaken together, our results suggest that miR-34a inhibits invasion and migration by targeting Smad4 to suppress EMT through TGF- beta/Smad signaling pathway in human EHCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1359-x) contains supplementary material, which is available to authorized users.

Significance of forkhead/winged helix transcription factor P3+ regulatory T cells infiltration in extrahepatic cholangiocarcinoma

Objective To explore the infiltration of regulatory T cells(Tregs)and its significance in extrahepatic cholangiocarcinoma(EHCC)and to elucidate the putative effects of cytochrome C oxidase- 2 (COX- 2)expression on the infiltration of Tregs. Methods The infiltration of forkhead/winged helix transcription factor P3(Foxp3)+ Tregs and the expression of COX- 2 were studied by immunohistochemistry in 94 and 77 cases of EHCC respectively.The correlation between the expression of COX- 2 and Foxp3+ Tregs infiltration with clinicopathological features and prognosis was analyzed. The influence of COX- 2 expression on infiltration of Foxp3+ Tregs was evaluated. Results Foxp3+ Tregs infiltration was seen in all the 94 cases of EHCC with the median Foxp3+ Tregs number being 2.35 per high- power field(HPF). There was no significant correlation between the number of infiltrated Foxp3+ Tregs in cancer tissue and histological grading, TNM stages and lymph node metastasis of EHCC.The patients with less number of infiltrated Foxp3+ Tregs showed significantly longer survival than those with greater number of infiltrated Foxp3+ Tregs(P<0.05).Cox analysis results showed that infiltration of Foxp3+ Tregs was an independent prognostic factor of EHCC(P< 0.05).The positive expression rate of COX- 2 in 77 cases of EHCC was 81.8%.COX- 2 expression was not significantly associatied with histological grading, TNM stages and lymph node metastasis of EHCC.COX- 2 expression was positively related with stromal Tregs infiltration(r= 4.89,P< 0.05). Conclusion The number of infiltrated Foxp3+ Tregs in EHCC tissue is negatively related with overall survival of the patients and is an independent prognosis factor of EHCC.COX-2 protein expression may contribute to Foxp3+ Tregs infiltration in cancer stroma. Key words: Extrahepatic cholangiocarcinoma; Cytochrome C oxidase-2; Forkhead/winged helix transcription factor P3; Regulatory T cells; Prognosis

Abnormal expression of Forkhead Box J2 (FOXJ2) suppresses migration and invasion in extrahepatic cholangiocarcinoma and is associated with prognosis.

Extrahepatic cholangiocarcinoma (CC) is an aggressive malignancy with dismal prognosis and characterized by early invasion, metastasis and postoperative recurrence. Therefore, understanding the main molecular mechanisms of this malignancy is the key for the development of novel and effective therapeutic strategies for extrahepatic CC. Foxj2 is a novel forkhead factor. Several FOX family members have been reported to play an important role in tumorigenesis and the progression of certain cancers. In this study, real-time quantitative RT-PCR (qRT-PCR), western blotting, and immunohistochemical staining were used to examine FOXJ2 expression in extrahepatic CC tissues and adjacent normal bile duct tissues. The molecular mechanisms of FOXJ2 expression and its effects on cell proliferation, migration and invasion were also explored by MTT assay, wound healing assay and Transwell assay. The relationships between the FOXJ2 expression levels, the clinicopathological factors, and patient survival were investigated. FOXJ2 mRNA and protein levels were downregulated in extrahepatic CC tissues compared to adjacent normal bile duct tissues. In addition, decreased FOXJ2 was associated disease progression in extrahepatic CC samples. Overexpression FOXJ2 expression markedly inhibited cell proliferation, migration and invasion in vitro. FOXJ2 is a transcription factor that has been reported to induce epithelial-mesenchymal transition (EMT). These findings indicated that FOXJ2 gene played a tumor suppressor role in extrahepatic CC, which proposed this gene as a new therapeutic target for extrahepatic CC patients.

Expression of Lamininγ2 in extrahepatic cholangiocarcinoma tissues and its influence on tumor invasion and metastasis.

To explore the expression of lamininγ2 in extrahepatic cholangiocarcinoma (EHCC) tissues and its influence on tumor invasion and metastasis. Paraffin embedding samples of cancer, para-cancer, lymph node metastatic and hepatic metastatic tissues from 79 patients undergoing EHCC resection were collected. Expression of lamininγ2 was detected by immunohistochemistry and its relationship with clinical pathological characteristics and the prognosis of EHCC patients were analyzed. Lamininγ2 showed negative staining in para-cancer tissues, but demonstrated a 51.9% (41/79) positive expression rate in extracellular matrix (ECM) or cytoplasm of EHCC tissues. In lymph node metastatic and distant metastatic nidi, expression of lamininγ2 was significantly higher than in the primary nidi (χ2=7.4173, P=0.0065; χ2=4.0077, P=0.0453). The expression was in obvious association with lymph node metastasis (P<0.01), but had no relevance with age, gender, tumor location, tumor stage, differentiation and distant metastasis in ECM (P>0.05), whereas it was in marked connection with lymph node and distant metastasis (P<0.05 or P<0.01), but had no relationship with age, gender, tumor location, tumor stage and differentiation in cytoplasm (P>0.05). However, the median survival time and median recurrent period of patients with positive expression of lamininγ2 in both cytoplasm and ECM of tumor cells, only in ECM and only in cytoplasm, were evidently lower than with negative expression of lamininγ2 in RCM and cytoplasm (P<0.05 or P<0.01). Further Cox regression analysis showed that the positive expression of lamininγ2 and the tumor differentiation were independent risk factors influencing the prognosis of EHCC patients. Abnormal expression of lamininγ2 may be closely associated with invasion and metastasis of tumor cells, and thus a potential molecular marker for prognosis of EHCC patients.

The role of nuclear EpICD in extrahepatic cholangiocarcinoma: association with β-catenin.

After intramembranous proteolysis-mediated loss of the extracellular domain of the epithelial cell adhesion molecule (EpEx) and release of an intracellular domain (EpICD) into the cytoplasm, EpICD sequentially associates with FHL2 to form a nuclear complex with β-catenin and Lef-1. This association induces gene transcription involved in the activation of the oncogenic potential of epithelial cell adhesion molecule (EpCAM). We examined the localization and expression of EpEx, EpICD and β-catenin in surgical specimens of extrahepatic cholangiocarcinoma (ECC) from 79 patients and focused on the relationship between nuclear expression of EpICD and β-catenin. We also examined the role of EpICD by transfecting the EpICD cDNA in cholangiocarcinoma (CC) cell lines. There was a significant correlation between the nuclear expression of EpICD and β-catenin in ECC tissues. Frequent nuclear co-localization of EpICD and β-catenin was observed in cancer cells forming the invasive front. Nuclear expression of EpICD also significantly correlated with histologic grade of tumor. Overexpression of EpICD in the CC cells significantly increased the cell growth and proliferation. The overexpression of EpICD in the CC cells also increased the expression levels of the active form of β-catenin and EpCAM target genes, such as c-myc and cyclinD1. Furthermore, the overexpression of EpICD significantly enhanced the migration and invasiveness of CC cells. Conversely, the inhibition of EpCAM in EpCAM-overexpressing cells by siRNA significantly decreased cell proliferation, migration and invasion. These results indicate that the spatial localization of EpICD and its mutual interaction with β-catenin may be important in ECC progression and invasion.

The role of nuclear EpICD in extrahepatic cholangiocarcinoma: association with beta-catenin

After intramembranous proteolysis-mediated loss of the extracellular domain of the epithelial cell adhesion molecule (EpEx) and release of an intracellular domain (EpICD) into the cytoplasm, EpICD sequentially associates with FHL2 to form a nuclear complex with beta-catenin and Lef-1. This association induces gene transcription involved in the activation of the oncogenic potential of epithelial cell adhesion molecule (EpCAM). We examined the localization and expression of EpEx, EpICD and beta-catenin in surgical specimens of extrahepatic cholangiocarcinoma (ECC) from 79 patients. There was a significant correlation between the nuclear expression of EpICD and beta-catenin in ECC tissues. Frequent nuclear co-localization of EpICD and beta-cate-nin was observed in cancer cells forming the invasive front. Forced overexpression of EpICD in the CC cells significantly increased the cell growth and proliferation. The overexpression of EpICD in the CC cells also increased the expression levels of the active form of beta-catenin and EpCAM target genes. Furthermore, the over-expression of EpICD significantly enhanced the migration and invasiveness of CC cells. Conversely, inhibition of EpCAM by siRNA significantly decreased the CC cell proliferation, migration and invasion. These results indicate that spatial localization of EpICD and its mutual interaction with beta-catenin may be important in ECC progression and invasion.

Detection of hepatitis B virus DNA in paraffin-embedded intrahepatic and extrahepatic cholangiocarcinoma tissue in the northern Chinese population

This study explored the importance of hepatitis B virus infection in cholangiocarcinoma pathogenesis in northern China. The clinical data of 66 patients with cholangiocarcinoma were analyzed. The hepatitis B virus gene was amplified using nested polymerase chain reaction, and the hepatitis B virus-related antigen was detected using immunohistochemistry in formalin-fixed, paraffin-embedded tissue from patients with intrahepatic cholangiocarcinoma (n = 23) and extrahepatic cholangiocarcinoma (n = 43). Hepatitis B surface antigen seropositivity was found in 52.2% (12/23) of intrahepatic cholangiocarcinoma cases and 13.9% (6/43) of extrahepatic cholangiocarcinoma cases. Hepatitis B virus DNA (X region) was detectable in 34.8% (8/23) of intrahepatic cholangiocarcinoma cases. Hepatitis B surface antigen and/or hepatitis B core antigen was detectable in 30.4% (7/23) of intrahepatic cholangiocarcinoma cases. All cases with detected viral protein were also positive for hepatitis B virus DNA. In contrast, no hepatitis B virus antigens or hepatitis B virus gene was detected in any of the 43 extrahepatic cholangiocarcinoma cases. Our findings strongly suggest that chronic hepatitis B virus infection is a significant risk factor for intrahepatic cholangiocarcinoma, but not for extrahepatic cholangiocarcinoma, in northern China. Hepatitis B virus infection is potentially independently associated with intrahepatic cholangiocarcinoma.

Isolation and characterization of tumorigenic extrahepatic cholangiocarcinoma cells with stem cell‐like properties

Recent studies suggest that the ability to form and grow tumors specifically resides in a small cell population called cancer stem cells (CSCs). These studies were conducted mainly on various human cancers; however, isolation and characterization of stem cells from cholangiocarcinoma have not been attempted. The molecular markers CD24, CD44, CD34, and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of CSCs. In this study, we used these markers to identify a subpopulation of cells in extrahepatic cholangiocarcinoma (ECC) with cancer stem/progenitor cell-like properties. We found that CD24(+) CD44(+) EpCAM(high) cells (0.39-2.27%) were present in human ECC tissues. The expression of a CD24(+) CD44(+) EpCAM(high) subpopulation was consistent with primary cancers and could be duplicated during serial in vivo passaging in NOD/SCID mice. CD24(+) CD44(+) EpCAM(high) cells isolated from 3 cholangiocarcinoma xenografts showed high tumorigenic potential compared with CD24(-) CD44(-) EpCAM(low/-) cells. These tumorigenic ECC cells exhibited the stem cell properties of self-renewal and ability to produce heterogeneous progeny. We report the identification of a CSC population in ECC characterized by CD24, CD44 and EpCAM phenotypes. Our findings could provide new insight into the tumorigenesis of cholangiocarcinoma and offer a potential target for anti-cancer therapy.

Human G-protein gamma 7 in extrahepatic cholangiocarcinoma and its clinicopathological significance

Several studies have found a down-regulated G-gamma 7 gene in gastrointestinal tract cancers. We evaluated the expression and clinicopathological significance of the human G protein gamma 7 (G-gamma 7) in human extra-hepatic cholangiocarcinoma (EHCC). The expression of G-gamma 7 expression was studied in 21 patients with EHCC. G-gamma 7 mRNA expression was tested by using RealTime reverse transcription polymerase chain reaction (RT-PCR).To visualize the localization of G-gamma 7, an immunohistochemistry study was also performed. The G-gamma 7 expression was compared among cancer tissues, peri-cancerous bile duct tissues and normal bile duct tissues. The clinicopathological significance of G-gamma 7 expression was also studied. Expression of G-gamma 7 mRNA and protein were significantly lower in EHCC tissue than in pericancerous bile duct tissue and normal bile duct tissues. G-gamma 7 mRNA and protein expression were significantly lower in poorly differentiated EHCC tissues than in moderate differentiated and well differentiated EHCC tissues (P<.01). There was no significant correlation between G-gamma 7 expression and host factors such as age, gender, clinical staging or the status of preoperative hepatic function EHCC has a down-regulated expression of G-gamma 7. Reduced expression of G-gamma 7 is associated with the histological grade of EHCC and may prove to be a useful marker for predicting the prognosis of human EHCC.

Loss of WWOX expression in human extrahepatic cholangiocarcinoma

WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that maps to the common fragile site FRA16D on chromosome 16q23.3-24.1. To investigate the role of the WWOX gene in the development of extrahepatic cholangiocarcinoma (ECC), 30 tissue samples of ECC were examined. Loss of heterozygosity (LOH), real time reverse transcription PCR (RT-PCR), and immunohistochemistry were used to assess the role of WWOX in cholangiocarcinoma. LOH was observed in 50% of cases, loss of mRNA was observed in 46.67% of the cases and loss of WWOX protein expression was found in 56.67% of ECC tissue samples. LOH, mRNA, and protein expression had a significant correlation with histological grading. The correlation coefficients were -0.623, -0.475 and -0.543, respectively. However, WWOX expression had no correlations with other clinicopathological factors such as age, gender, clinical staging or the status of preoperative hepatic function (p > 0.05). Poorly differentiated ECC had significantly lower expression of WWOX than that of moderately or well differentiated ones (p < 0.05). Loss of WWOX may be involved in the tumorigenesis of extrahepati cholangiocarcinoma.

Correlation of aPKC-t and E-cadherin expression with invasion and prognosis of extrahepatic cholangiocarcinoma

Objective To investigate the correlation of aPKC-t and E-cadherin expression with clinicopathological characteristics and prognosis in extrahepatic cholangiocarcinoma and analyze molecular mechanisms of invasion and metastasis in extrahepatic cholangiocarcinoma. Methods EnVision immunohistochemistry was applied to defect the expression of aPKC-t and E-cadherin in 9 specimens of benign bile duct tissues, 35 of extrahepatic cholangiocarcinoma and 6 of metastatic carcinoma of cholangiocarcinoma. The relation of the expression to clinicopathological characteristics, invasion and prognosis of extrahepatic cholangiocarcinoma was analyzed. A multivariate regression analysis was performed in these patients by the computer＇s Cox proportional hazard model. Results The positive expression intensity of aPKC-t in extrahepatic cholangiocarcinoma was remarkably stronger than that in benign bile duct tissues, while the expression intensity of E-cadherin was weaker in extrahepatic cholangiocarcinoma than that in benign bile duct tissues. Correlation analysis revealed that the expression of aPKC-t was positively related to the progress of the tumor pathological differentiation and invasion, whereas that of E-cadherin was entirely contrary. Moreover, there was a negative relationship between the expression of aPKC-t and that of E-cadherin（r=0. 387,P〈0. 05）. The univariate analysis showed that all survival rates of the group with higher aPKC-t expression in extrahepatic cholangiocarcinoma was remarkably less than that in the group with lower expression （P〈0. 01）. Multivariate analysis revealed that the expression of aPKC-t and E-cadherin was the most important prognostic factor for extrahepatic cholangiocarcinoma （P〈0.05）. Conclusion The expression of aPKC-t and E-cadherin may reflect the differentiation and invasive potential of extrahepatic cholangiocarcinoma tissues to varying degrees, aPKC-t and E-cadherin may be independent prognostic factors and, when used in combination with clinicopathological characteristics, may increase accuracy of predicting prognosis of patients with extrahepatic cholangiocarcinoma. As a polar regulative associated protein, aPKC-t may play an important role in the invasion and metastasis of extrahepatic cholangiocarcinoma. Key words: Biliary tract neoplasms; Atypical protein kinase C; E-cadherin; Immunohistochemistry