Biomarker For Cholangiocarcinoma Research Articles

Quantitative Proteomic Analysis and Evaluation of the Potential Prognostic Biomarkers in Cholangiocarcinoma.

Background & Aims: Cholangiocarcinoma (CCA) patients have poor outcomes since the late diagnosis limits the benefits of surgery therapy and curative treatment options. The present study was designed to screen the biomarkers for CCA patients.Methods: Quantitative iTRAQ proteomic analysis was used to identify differentially expressed proteins between CCA and pericarcineous tissue. We examined the expression profile of PRDX2, BGN, LUM, and PPP3CA in CCA tissue using immunohistochemistry. We further investigated the correlation between PPP3CA expression and the survival of CCA patients (n=91).Results: 2,886 confidential proteins were identified by using the iTRAQ technique, 233 of which were differentially expressed. PRDX2, BGN, PPP3CA, and LUM were expressed in CCA tissue, whereas they were not expressed in choledocal cyst tissue except for LUM. PPP3CA was expressed in the cytoplasm of carcinoma cells in 22 cases (24.2%) of 91 CCA patients. Patients with PPP3CA-positive expression showed a significantly shorter survival period than did non-expressing patients (P = 0.030). The univariate analysis showed that tumor size (P = 0.002), vascular invasion (P = 0.001), histological grading (P = 0.011), and PPP3CA expression (P = 0.033) were statistically significant risk factors affecting the prognosis of CCA patients. The multivariate analysis demonstrated PPP3CA expression (P = 0.009) and vascular invasion (P = 0.012) were statistically significant independent risk factors of CCA patients.Conclusions: The results suggested that the expression of PPP3CA in CCA patients is a new independent factor for poor prognosis and a useful prognostic predictor for patients with CCA.

Prognostic Significance of Platelet-to-Lymphocyte Ratio in Cholangiocarcinoma: A Meta-Analysis.

Introduction Pretreatment platelet-to-lymphocyte ratio (PLR) has been considered a prognostic factor in various cancers. However, the application of PLR in the assessment of patients with cholangiocarcinoma remains controversial. This study aimed to evaluate the prognostic value of pretreatment PLR in cholangiocarcinoma. Methods A systematic search was performed in MEDLINE, EMBASE, and Cochrane Library to identify studies assessing the prognostic significance of the pretreatment PLR in cholangiocarcinoma. Three databases were searched from inception to August 5, 2018. The primary outcome was overall survival (OS), and the secondary outcomes were recurrence-free survival (RFS) and progression-free survival (PFS). Pooled hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using random-effects models. Results A total of 9 studies including 2395 patients were finally enrolled in the meta-analysis based on the inclusion and exclusion criteria. All of the included studies were retrospective observational cohorts. Elevated PLR predicted poor OS (HR: 1.38, 95% CI: 1.19-1.62, P < 0.001) and RFS or PFS (HR = 1.55; 95% CI = 1.27-1.88; P < 0.001). Moreover, elevated PLR was highly associated with male sex (male versus female OR = 0.59, 95% CI: 0.44-0.80, P < 0.001) and R1 resection margin (OR = 2.09, 95% CI: 1.24-3.54, P = 0.006). Conclusion The present meta-analysis demonstrated that pretreatment PLR might serve as a useful prognostic biomarker in cholangiocarcinoma.

Discovery of glycocholic acid and taurochenodeoxycholic acid as phenotypic biomarkers in cholangiocarcinoma

Although several biomarkers can be used to distinguish cholangiocarcinoma (CCA) from healthy controls, differentiating the disease from benign biliary disease (BBD) or pancreatic cancer (PC) is a challenge. CCA biomarkers are associated with low specificity or have not been validated in relation to the biological effects of CCA. In this study, we quantitatively analyzed 15 biliary bile acids in CCA (n = 30), BBD (n = 57) and PC (n = 17) patients and discovered glycocholic acid (GCA) and taurochenodeoxycholic acid (TCDCA) as specific CCA biomarkers. Firstly, we showed that the average concentration of total biliary bile acids in CCA patients was quantitatively less than in other patient groups. In addition, the average composition ratio of primary bile acids and conjugated bile acids in CCA patients was the highest in all patient groups. The average composition ratio of GCA (35.6%) in CCA patients was significantly higher than in other patient groups. Conversely, the average composition ratio of TCDCA (13.8%) in CCA patients was significantly lower in all patient groups. To verify the biological effects of GCA and TCDCA, we analyzed the gene expression of bile acid receptors associated with the development of CCA in a CCA cell line. The gene expression of transmembrane G protein coupled receptor (TGR5) and sphingosine 1-phosphate receptor 2 (S1PR2) in CCA cells treated with GCA was 8.6-fold and 3.4-fold higher compared with control (untreated with bile acids), respectively. Gene expression of TGR5 and S1PR2 in TCDCA-treated cells was not significantly different from the control. Taken together, our study identified GCA and TCDCA as phenotype-specific biomarkers for CCA.

Angiogenic miRNAs, the angiopoietin axis and related TIE2-expressing monocytes affect outcomes in cholangiocarcinoma.

BackgroundTumour angiogenesis is modulated on both an epigenetic and protein level and has potential implications for immune cell responses. However, the importance of related angiogenic biomarkers in cholangiocarcinoma (CCA) is unknown. This study assessed human CCA samples for the expression of angiogenesis-associated microRNAs, angiopoietins (Angs) and monocytes expressing the Ang-receptor, TIE2, with regards to prognostic significance after liver resection.MethodsAngiogenic miRNAs were analysed in frozen samples of intrahepatic CCA (iCC; n = 43) and hilar CCA (HC; n = 45). Ang-1 and Ang-2, as well as TIE2-expressing monocytes (TEMs), were detected in paraffin-embedded iCC sections (n = 88). MiRNA expression and the abundance of TEMs and Angs were correlated with clinicopathological characteristics and survival.ResultsMiR-126 was downregulated in 76.7% of all CCA samples, with high relative expression associated with smaller tumours and reduced lymph node metastasis. High Ang-1 expression was associated with less lymphangiosis carcinomatosa and better histological grading (all p < 0.05). The absence of TEMs in iCC correlated with elevated CA19-9 levels. High relative miR-126 and low miR-128 levels were associated with improved survival in iCC and HC, respectively (all p < 0.05). High miR-126, low miR-128 and TEMs were independent prognostic factors for recurrence-free and overall survival (all p < 0.05).ConclusionsThese results suggest that angiogenic miRNAs, Angs and TEMs are of prognostic value in CCA. In addition to the possible functional links between angiogenic miRNA expression profiles, Angs and immune-cell responses by TEMs, these data have clinical implications as novel diagnostic tools.

Proteomics detection of S100A6 in tumor tissue interstitial fluid and evaluation of its potential as a biomarker of cholangiocarcinoma.

Tumor interstitial fluid contains tumor-specific proteins that may be useful biomarkers for cancers. In this study, we identified proteins present in cholangiocarcinoma interstitial fluid. Proteins derived from three samples of tumor interstitial fluid and paired samples of adjacent normal interstitial fluid from cholangiocarcinoma patients were subjected to two-dimensional liquid chromatography with tandem mass spectrometry. Candidate proteins were selected based on a greater than twofold change in expression levels between tumor interstitial fluid and normal interstitial fluid. Upregulation of six proteins in tumor interstitial fluid, including S100 calcium binding protein A6 (S100A6), S100 calcium binding protein A9, aldo-keto reductase family 1 member C4, neuropilin-1, 14-3-3 zeta/delta, and triosephosphate isomerase was assessed by western blot and immunohistochemistry. Their potential as markers was evaluated in human cholangiocarcinoma tissue arrays, and in serum using enzyme-linked immunosorbent assay. Expression of S100A6 was higher in tumor interstitial fluid than in normal interstitial fluid and showed the highest positive rate (98.96%) in cholangiocarcinoma tissues. Serum levels of S100A6 did not differ between cholangitis and cholangiocarcinoma patients, but were significantly higher than in healthy individuals ( p < 0.0001). In cholangiocarcinoma cases, S100A6 level was associated with vascular invasion ( p = 0.007) and could distinguish cholangiocarcinoma patients from healthy individuals as effectively as the carbohydrate antigen 19-9. In addition, potential for drug treatment targeting S100A6 and other candidate proteins was also demonstrated using STITCH analysis. In conclusion, proteomics analysis of tumor interstitial fluid could be a new approach for biomarker discovery, and S100A6 is a potential risk marker for screening of cholangiocarcinoma.

Expression and Serum Levels of Mucin 5AC (MUC5AC) as a Biomarker for Cholangiocarcinoma: a Meta-analysis.

The potential of biomarkers in detecting early cholangiocarcinoma (CCA) is facilitated by examining CCA-associated proteins from primary studies. One such protein is mucin 5AC (MUC5AC) but inconsistency of reported associations between its expression/serum levels and CCA prompts a meta-analysis to obtain more precise estimates. A literature search yielded 17 included articles where multiple data in some raised the number of studies to 22. We calculated pooled odds ratios (OR) and 95% confidence intervals from negative and positive readings of MUC5AC levels. Data were subgrouped by ethnicity, detection method, sample source, and cancer type. Outcome in the overall analysis was non-significant but those in the subgroups were. Thus, significant associations (P < 0.001) indicating high MUC5AC levels were found in three subgroups: (i) Thai (OR 8.32) and (ii) serum (OR 4.52). Heterogeneity of these two outcomes (I2 = 90-93%) was erased with outlier treatment (I2 = 0%) which also modulated the pooled effects (OR 2.48-2.59). (iii) Immunoblot (OR 2.61) had low initial heterogeneity (I2 = 2%). Robustness and significant tests for interaction (Pinteraction = 0.01-0.02) improved MUC5AC associations with CCA in the Thai population. Our pooled effect findings target the biomarker potential of MUC5AC to the Thai population.

MiRNA and lncRNA as biomarkers in cholangiocarcinoma(CCA).

The microRNAs are a group of 20 nucleotides-long non-coding RNAs. By binding to the 3’UTR region of target mRNA, microRNAs can perform extensive actions mediating gene expression at post-trancriptional stages. It makes microRNAs serve as very crucial regulators in various biological progress including carcinogenesis. Long non-coding RNAs, however, are a subgroup of RNA with the length of 200 nucleotides. Unlike microRNAs, long non-coding RNAs can form secondary of tertiary domain based on their length. With the ability of directly interacting with DNA, RNA, protein, long non-coding RNAs have promoting or inhibitive functions in gene expression regulation. Furthermore, the abnormal expression of certain long non-coding RNAs has roused people’s interest in the role of long non-coding RNAs in tumorigenesis. Although the connection between microRNA/long non-coding RNA and CCA has been a hot field to researchers, the link between molecular mechanism and clinical outcome has been barely built. This review takes a retrospect at the latest researches on the link between microRNA/long non-coding RNA and cholangiocarcinoma and the potential of microRNA/long non-coding RNA serving as distinctive biomarkers for CCA in clinical practice.

MicroRNAs: the emerging regulatory molecules and biomarkers of cholangiocarcinoma

MicroRNAs (miRNAs) are closely associated with the development, invasion, metastasis and prognosis of cholangiocarcinoma (CCA). The abnormal expressions of miRNA play important roles in regulating the genetic variation, cell cycle, invasion and metastasisability and apoptosis of CCA. MiRNAs are hopeful for being used as the biological markers of early diagnosis, prognosis and treatment target. Key words: Bile duct neoplasms; MicroRNAs; Biological markers

Diagnostic value of microRNAs as biomarkers for cholangiocarcinoma

BackgroundIt has been reported that microRNAs might serve as biomarkers for cholangiocarcinoma (CCA) detection, but their diagnostic accuracy are unclear and controversial. AimTo evaluate the diagnostic accuracy of microRNAs for CCA. Materials and methodsPubMed, Embase, Web of Science and Cochrane Library were systematically searched to identify relevant articles by using the key words “cholangiocarcinoma” and “microRNA”. The methodological quality of each study was assessed by QUADAS-2. According to the inclusive and exclusive criteria, 11 articles were identified and analyzed by Meta-disc software v.1.4 and STATA 12.0 software package. Results11 articles with 430 CCA patients and 406 controls were identified. The results showed that the pooled sensitivity was 0.756 (95% CI: 0.693–0.810), specificity was 0.914 (95% CI: 0.861–0.947), positive likelihood ratio was 8.747 (95% CI: 5.320–14.381), negative likelihood ratio was 0.267 (95% CI: 0.208–0.341) and diagnostic odds ratio was 32.791 (95% CI: 17.327, 62.060). In addition, the area under the summary ROC cure (AUC) was 0.90 (95% CI: 0.87–0.93). ConclusionMicroRNAs have great potential as biomarkers for the diagnosis of CCA patients. However, the clinical application of microRNA assays for CCA diagnosis still needs further validation by more prospective studies.

Abstract A37: Aberrant DNA methylation of HTATIP2 and UCH-L1 as prognostic and predictive biomarkers for cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a malignancy of bile duct epithelial cell lining. In the past decade, the incidence and mortality rates of CCA have been increasing worldwide. The effective treatment modality for CCA is surgical resection. Adjuvant chemotherapy and/or radiotherapy are optional therapy for unresectable and postoperative CCA. Unfortunately, the responsiveness of CCA patients to chemotherapy was relatively low and 5-year survival rate was poor. We have analyzed data which were obtained from our previous study on methylation microarray based on chemotherapeutic drug resistance and found aberrant DNA methylation of genes related to chemotherapy. This present study aimed to quantitate DNA methylation levels of 10 candidate genes related to chemotherapy in 54 CCA and 19 normal adjacent tissues using methylation-sensitive high resolution melting (MS-HRM) analysis. The methylation level of each gene was statistically analyzed with clinical parameters. Genes with high methylation level were UCH-L1 and IRF4, and genes with moderate methylation were CCND2, HTATIP2 and TP53I3. We found significant difference in methylation levels of HTATIP2 (P=0.036) and UCH-L1 (P=0.012) between short and long survival groups. Patients with high methylation level of HTATIP2 and low methylation of UCH-L1 showed longer overall survival (P=0.03 and P=0.044, respectively). Interestingly, chemotherapeutic responders showed higher methylation level of HTATIP2 than non-responders (P=0.020), whereas no significant difference in UCH-L1 methylation level was observed between these two groups. The methylation levels of HTATIP2 and UCH-L1 were low in normal adjacent tissues. In patients who received chemotherapy, patients who had high methylation of HTATIP2 and low methylation of UCH-L1 showed longer overall survival (P&amp;lt;0.001 and P=0.041, respectively). Moreover, HTATIP2 methylation was associated with tumor progression in which lower methylation level was found in CCA patients with positive lymph node metastasis (P=0.029). In conclusion, our finding indicates the great value of DNA methylation levels of HTATIP2 and UCH-L1 as prognostic and predictive markers for CCA patients. Citation Format: Temduang Limpaiboon, Chaiyachet Nanok, Siriporn Proungvitaya, Patcharee Jearanaikoon, Ake Pugkhem, Anucha Puapairoj. Aberrant DNA methylation of HTATIP2 and UCH-L1 as prognostic and predictive biomarkers for cholangiocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A37.

Serum miR-26a as a diagnostic and prognostic biomarker in cholangiocarcinoma.

In order to determine the diagnostic and prognostic value of miR-26a in Cholangiocarcinoma (CCA), we compared miR-26a levels in serum from 66 CCA patients and 66 healthy controls, which was followed by serum analysis between the pre-operative serum and post-operative serum of these CCA patients. We found the concentration levels of miR-26a in serum of CCA patients were significantly higher than that from healthy controls (P < 0.01). Furthermore, the concentration levels of miR-26a in the post-operative serum were significantly reduced when compared to the pre-operative serum (P < 0.001). High miR-26a in serum was correlated significantly with clinical stage, distant metastasis, differentiation status, and poor survival of CCA patients. More importantly, serum miR-26a was an independent prognostic marker for CCA. In conclusion, our results suggested that miR-26a in serum might be a potential and useful noninvasive biomarker for the early detection of CCA.

412: DNA methylation levels of OPCML and SFRP1 as diagnostic biomarkers in cholangiocarcinoma

412: DNA methylation levels of OPCML and SFRP1 as diagnostic biomarkers in cholangiocarcinoma

Tumoral heterogeneity of hepatic cholangiocarcinomas revealed by MALDI imaging mass spectrometry

Cholangiocarcinoma (CC) is the second most common primary malignancy of the liver. Although all CC derive from biliary epithelial cells, two main subtypes, hilar (H), and peripheral (P) CC are described. The objective of the study was to compare, using MALDI imaging mass spectrometry (MALDI IMS), in situ proteomic profiles of H- and P-CC in order to assess whether these subtypes may express different markers and to describe their respective localizations. Twenty-seven CC (16 P-CC and 11 H-CC) were subjected to MALDI IMS. Proteomic data were submitted to a dedicated cross-classification comparative design, enabling comparison of the entire generated spectra. Immunohistochemistry was performed for validation. Comparative analysis yielded a list of 19 differential protein peaks for the two subtypes, 14 of which were overexpressed in H-CC and five in P-CC. Among H-CC protein markers, most discriminant were human neutrophil peptides 1-3 that were expressed mainly by tumor cells and S100 proteins (A6 and A11) that were restricted to the stromal area. In P-CC, thymosin β4 was diffusely overexpressed. These results highlight the potential of MALDI IMS to discover new relevant biomarkers of CC and to characterize the heterogeneity of the two different subtypes.

FXYD6 is a new biomarker of cholangiocarcinoma

Members of the FXYD domain-containing ion transport regulator protein family, including FXYD3 and FXYD5, play an important role in the pathogenesis of numerous tumors. However, the correlation between the expression of FXYD6 and tumors remains poorly understood. In the current study, the expression of FXYD6 was examined immunohistochemically in 72 cholangiocarcinoma tissues and 30 distal normal bile duct tissues matched with the tumors. The results show that the positive expression rate of FXYD6 was significantly higher in cholangiocarcinoma than that in normal bile duct tissue (69 vs. 33.3%; P=0.002). Furthermore, the positive expression rate of FXYD6 in well- and moderately-differentiated cholangiocarcinoma was clearly higher than that in poorly-differentiated and mucinous cholangiocarcinoma (85.7 vs. 40%; P=0.000). However, there was no significant correlation between the expression of FXYD6 and gender (P=0.393), age (P=0.174), histological type (P=0.123), T stage (P=0.164), lymph node metastasis (P=0.343), perineural invasion (P=0.088) and tumor location (P=0.238). The results of this study indicate that FXYD6 may be a new biomarker for cholangiocarcinoma and may be associated with a favorable prognosis in this malignant disease.

Role of biliary CEACAM6 as a biomarker for cholangiocarcinoma.

177 Background: Distinguishing bile duct carcinoma from other diagnoses is often difficult using endoscopic or percutaneous techniques. The cell surface protein CEACAM6 is over-expressed in many gastrointestinal cancers and may be selectively elevated in biliary adenocarcinoma. The aim of the present study is to determine if CEACAM6 can be detected in the bile of patients with biliary cancer and can serve as a diagnostic biomarker for cholangiocarcinoma. Methods: Bile duct and gallbladder bile from patients with benign biliary disease and cholangiocarcinoma (hilar, intrahepatic and distal) was collected at the time of index operation. The concentration of CEACAM6 was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) and correlated to pathologic diagnosis. Diagnostic capability of CEACAM6 was evaluated by Wilcoxon rank-sum, logistic regression, and receiver operating characterisitic (ROC) curve analysis. Results: Bile from 73 patients was analyzed: 40 with benign disease and 33 with cholangiocarcinoma. Patients in the benign cohort were younger, predominantly female, and had lower mean biliary CEACAM6 levels than patients in the malignant cohort (102 ng/ml vs. 239 ng/ml; p=0.0006). Logistic regression analysis determined CEACAM6 to be a positive predictor of extrahepatic but not intrahepatic cholangiocarcinoma (p=0.0015). ROC curve with a CEACAM6 level &gt;22.1 ng/ml was associated with 85% sensitivity, 73% specificity, and a likelihood ratio of 3.15 (AUC 0.82) for extrahepatic cholangiocarcinoma. Conclusions: CEACAM6 levels in bile can predict patients with extrahepatic cholangiocarcinoma with acceptable accuracy. Further investigation is warranted in a larger cohort of patients prior to clinical application.

Comparative Proteomic Profiling of Human Bile Reveals SSP411 as a Novel Biomarker of Cholangiocarcinoma

BackgroundCholangiocarcinoma (CC) is an intractable cancer, arising from biliary epithelial cells, which has a poor prognosis and is increasing in incidence. Early diagnosis of CC is essential as surgical resection remains the only effective therapy. The purpose of this study was to identify improved biomarkers to facilitate early diagnosis and prognostication in CC.MethodsA comparative expression profile of human bile samples from patients with cholangitis and CC was constructed using a classic 2D/MS/MS strategy and the expression of selected proteins was confirmed by Western blotting. Immunohistochemistry was performed to determine the expression levels of selected candidate biomarkers in CC and matched normal tissues. Finally, spermatogenesis associated 20 (SSP411; also named SPATA20) was quantified in serum samples using an ELISA.ResultsWe identified 97 differentially expressed protein spots, corresponding to 49 different genes, of which 38 were upregulated in bile from CC patients. Western blotting confirmed that phosphoglycerate mutase 1 (brain) (PGAM-1), protein disulfide isomerase family A, member 3 (PDIA3), heat shock 60 kDa protein 1 (chaperonin) (HSPD1) and SSP411 were significantly upregulated in individual bile samples from CC patients. Immunohistochemistry demonstrated these proteins were also overexpressed in CC, relative to normal tissues. SSP411 displayed value as a potential serum diagnostic biomarker for CC, with a sensitivity of 90.0% and specificity of 83.3% at a cutoff value of 0.63.ConclusionsWe successfully constructed a proteomic profile of CC bile proteins, providing a valuable pool novel of candidate biomarkers. SSP411 has potential as a biomarker for the diagnosis of CC.

318 INCREASED LEVELS OF NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN (NGAL) IN THE PLASMA OF CHOLANGIOCARCINOMA PATIENTS

Introduction We have previously demonstrated that the level of neutrophil gelatinase-associated lipocalin (NGAL) is increased in the bile of patients with pancreatobiliary malignancy. NGAL is expressed by activated neutrophils and many other cell types and is thought to have bacteriostatic, pro-proliferative and pro-metastatic functions. NGAL can be detected in the blood plasma. We hypothesised that the plasma NGAL level is elevated in patients with cholangiocarcinoma (CC) compared with patients with primary sclerosing cholangitis (PSC), and with healthy volunteers. Methods Plasma samples were collected from 97 patients with confirmed CC, 62 patients with PSC and no CC and 82 healthy controls. Plasma NGAL quantification was performed in duplicate on plasma from each subject using a Quantikine ELISA kit (R&D Systems, Minneapolis, Minnesota, USA). CC and healthy control cohorts were compared using the Student t test and receiver operator characteristic (ROC) analysis. Differences between CC and PSC cohorts were then sought. Pearson9s correlation analysis was used to assess relationships between the levels of NGAL and other plasma markers. Results Median NGAL concentrations (range) in CC, PSC and healthy controls were 92 ng/ml (14–644), 83 (43–171) and 64 (29–132) respectively. NGAL levels were significantly higher in plasma from CC patients compared with healthy controls (p 2 =0.14), white cell count (r 2 =0.09), bilirubin (r 2 =0.01), ALP (r 2 =0.02) or creatinine (r 2 =0.03). There was moderate correlation between NGAL and Ca19-9 concentrations (r 2 =0.38). Conclusion NGAL is expressed at significantly higher concentrations in the plasma of patients with CC compared to plasma from healthy controls and from subjects with PSC. This finding appears to be independent of renal impairment, cholestasis or systemic inflammatory response, suggesting that NGAL may represent a novel plasma biomarker of CC. Competing interests None declared.

Heat Shock Proteins 27 and 70 Are Potential Biliary Markers for the Detection of Cholangiocarcinoma

Cholangiocarcinoma often is diagnosed at an advanced stage. Thus, it is necessary to establish sensitive screening methods that would allow cholangiocarcinoma and preferably its precursor lesion [biliary intraepithelial neoplasia (BilIN)] to be detected. We sought to clarify the usefulness of heat shock protein (HSP) 27 and HSP70 as biomarkers of cholangiocarcinoma and have used immunohistochemical analyses of hepatolithiatic livers to characterize HSP27 and HSP70 expression during the multistep cholangiocarcinogenesis process. HSP27 and HSP70 were measured in serum and bile samples via enzyme-linked immunosorbent assay. In hepatolithiatic tissue, the expression of HSP27 and HSP70 was increased in BilIN as well as in invasive cholangiocarcinoma. The serum levels of HSP27 and HSP70 were not significantly different between the hepatolithiatic patients with and without cholangiocarcinoma. In contrast, the bile levels of HSP27 and HSP70 were increased significantly in the patients with cholangiocarcinoma compared with those in the patients with lithiasis. Combining the measurements of the bile levels of HSP27 and HSP70 increased their usefulness as biomarkers, and the sum (HSP27 + HSP70) yielded the best sensitivity (90%) and specificity (100%). These results suggest that HSP27 and HSP70 could be used as biliary biomarkers for the detection of cholangiocarcinoma including BilIN.

Identification of differentially expressed biliary proteins induced by cholangiocarcinoma using 2D-DIGE

Objective To determine the probability of identification of differential expression of biliary proteins induced by cholangiocarcinoma using 2D-DIGE. Methods Bile was obtained from 12patients with obstructive jaundice (including 6 cases of cholangiocarcinoma and 6 of cholelithiasis).Each sample was labeled with three different CyDyes (y3,Cy5,Cy2) including one internal standard,pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. MALDI-TOF-MS and bioinformatics were adopted to identify and elucidate the significance of differentially expressed proteins in bile induced by cholangiocarcinoma. Results 55 matched protein spots differences in abundance were detected with statistical variance of two groups(Average Volum Ratio ≥1.5, t-test, P＜0. 05). Among these proteins, 13 PMF were obtained by MALDI-TOF-MS analysis. Eight proteins were identified by searching a protein database. Conclusion The differentially displayed proteomes between the pathological bile obtained from benign and malignant obstructive jaundice indicates the potential application of 2D-DIGE to identify the biomarker of cholangiocarcinoma. Key words: Bile duct neoplasms; Bile; Fluorescence two-dimensional difference gel electrophoresis

Trefoil factors: Tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma

To investigate trefoil factor (TFF) gene copy number, mRNA and protein expression as potential biomarkers in cholangiocarcinoma (CCA). TFF mRNA levels, gene copy number and protein expression were determined respectively by quantitative reverse transcription polymerase chain reaction (PCR), quantitative PCR and immunohistochemistry in bile duct epithelium biopsies collected from individuals with CCA, precancerous bile duct dysplasia and from disease-free controls. The functional impact of recombinant human (rh)TFF2 peptide treatment on proliferation and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling was assessed in the CCA cell line, KMBC, by viable cell counting and immunoblotting, respectively. TFF1, TFF2 and TFF3 mRNA expression was significantly increased in CCA tissue compared to disease-free controls, and was unrelated to gene copy number. TFF1 immunoreactivity was strongly increased in both dysplasia and CCA, whereas TFF2 immunoreactivity was increased only in CCA compared to disease-free controls. By contrast, TFF3 immunoreactivity was moderately decreased in dysplasia and further decreased in CCA. Kaplan-Meier analysis found no association of TFF mRNA, protein and copy number with age, gender, histological subtype, and patient survival time. Treatment of KMBC cells with rhTFF2 stimulated proliferation, triggered phosphorylation of EGFR and downstream extracellular signal related kinase (ERK), whereas co-incubation with the EGFR tyrosine kinase inhibitor, PD153035, blocked rhTFF2-dependent proliferation and EGFR/ERK responses. TFF mRNA/protein expression is indicative of CCA tumor progression, but not predictive for histological sub-type or survival time. TFF2 is mitogenic in CCA via EGFR/MAPK activation.