Biliary Tract Carcinoma Cell Research Articles

Gamma-synuclein is a novel prognostic marker that promotes tumor cell migration in biliary tract carcinoma.

Gamma‐synuclein (SNCG) promotes invasive behavior and is reportedly a prognostic factor in a range of cancers. However, its role in biliary tract carcinoma (BTC) remains unknown. Consequently, we investigated the clinicopathological significance and function of SNCG in BTC. Using resected BTC specimens from 147 patients with adenocarcinoma (extrahepatic cholangiocarcinoma [ECC, n = 96]; intrahepatic cholangiocarcinoma [ICC, n = 51]), we immunohistochemically evaluated SNCG expression and investigated its correlation with clinicopathological factors and outcomes. Furthermore, cell lines with high SNCG expression were selected from 16 BTC cell lines and these underwent cell proliferation and migration assays by siRNAs. In the results, SNCG expression was present in 22 of 96 (22.9%) ECC patients and in 10 of 51 (19.6%) ICC patients. SNCG expression was significantly correlated with poorly differentiated tumor in both ECC and ICC (p = 0.01 and 0.03, respectively) and with perineural invasion and lymph node metastases in ECC (p = 0.04 and 0.003, respectively). Multivariate analyses revealed that SNCG expression was an independent poor prognostic factor in both OS and RFS in both ECC and ICC. In vitro analyses showed high SNCG expression in three BTC cell lines (NCC‐BD1, NCC‐BD3, and NCC‐CC6‐1). Functional analysis revealed that SNCG silencing could suppress cell migration in NCC‐BD1 and NCC‐CC6‐1 and downregulate cell proliferation in NCC‐CC6‐1 significantly. In conclusion, SNCG may promote tumor cell activity and is potentially a novel prognostic marker in BTC.

Gamma-synuclein as a potential novel prognostic marker promoting tumor cell migration in biliary tract carcinoma.

577 Background: Biliary tract carcinoma (BTC) is a highly malignant tumor, but the detailed pathological mechanism of its development and progression remain ill-defined. Gamma-synuclein (SNCG) promotes invasive behavior in pancreatic cancer and a range of other cancers, and SNCG expression is reportedly a prognostic factor. However, the role of SNCG in BTC remains unknown. Consequently, we investigated the clinicopathological significance and function of SNCG in BTC. Methods: Using surgically resected BTC specimens from 149 patients with adenocarcinoma [extrahepatic cholangiocarcinoma (ECC) (n =98); intrahepatic cholangiocarcinoma (ICC) (n =51)], we immunohistochemically evaluated SNCG positivity and checked for correlations with clinicopathological factors and outcomes. Furthermore, cell lines with high expressions of SNCG were selected from 17 BTC cell lines by using immunohistochemistry and qPCR. Cell proliferation and migration assays were then performed in the presence and absence of SNCG (silenced using). Results: SNCG expression was found in 32 of 149 (21.4%) BTC patients. SNCG expression was significantly correlated with poorly differentiated tumor ( P = 0.001) and lymph node metastases ( P = 0.001). SNCG positivity was also correlated with poorly differentiated tumor in both ECC and ICC ( P = 0.008 and P = 0.03, respectively), but was correlated with lymph node metastases only in ECC ( P = 0.003). Multivariate analyses identified SNCG expression as an independent prognostic factor of poor overall survival ( P = 0.008) and poor recurrence-free survival (RFS) ( P = 0.006). SNCG expression in both ECC and ICC was significantly associated with poor prognosis in univariate analysis, and multivariate analysis identified SNCG as an independent prognostic factor of poor RFS for ECC (P = 0.03). High SNCG expression was found in 3 BTC cell lines (NCC-BD1, NCC-BD3, and NCC-CC6-1). Functional analysis revealed that SNCG silencing by siRNA suppressed cell migration significantly in NCC-BD1 and NCC-CC6-1 and downregulated cell proliferation in NCC-CC6-1. Conclusions: Our results suggest that SNCG may promote tumor cell activity and is potentially a novel prognostic marker in BTC.

Abstract 1082: Loss of desmoglein 2 promotes tumor growth and progression through the activation of Src and facilitates the internalization of EGFR in biliary tract carcinoma cells

Abstract Understanding the molecular carcinogenesis pathways is critical to establish novel individualized molecular targeted therapies and finally to improve overall clinical outcomes of patients with a highly heterogeneous group of malignancies such as biliary tract cancers (BTCs). In this context, we investigated the patho-physiological roles of a desmosomal component, desmoglein 2 (Dsg2) and found that Dsg2 functions as a tumor suppressor protein in BTC, as Dsg2 loss induced a number of cellular biological activities including proliferation, motility, invasion, transendothelial migration in vitro and tumor growth and metastasis in vivo. Clinico-pathological analyses of BTC patients indicated that lower expression of Dsg2 is associated with a poor prognosis of patients as perineural and lymphatic invasion were highly increased in those patients. In addition, loss of Dsg2 showed a significant increase of EGFR clearance from the cell membrane in BTC cells and patients' tumor tissues. Those cells were significantly decreased in the EGF-driven activation of EGFR and highly resistant to the anti-EGFR antibody treatment. Src appears to be involved in the Dsg2-mediated clearance of EGFR, as this significantly blocked by the suppression of cSrc expression in Dsg2-depleted cells, possibly through the direct binding of Dsg2 preferentially with an inactive form of Src through its SH2 domain. Suppression of cSrc by Src inhibitors or siRNA dramatically reduced BTC tumor growth in vitro and in vivo. In comparison with the anti-EGFR antibody therapy, Src-targeted therapies produced consistent anti-tumor activities independent with the expression status of EGFR in the BTC tumors. In conclusion, targeting Src would be a promising therapeutic approach to patients with advanced BTC and potentially useful in overcoming resistance to current anti-EGFR therapies, although further clinical studies are essentially required to translate the current preclinical knowledge into clinical practice. Citation Format: Sang-Hyun Lee, Jeong-Ki Min. Loss of desmoglein 2 promotes tumor growth and progression through the activation of Src and facilitates the internalization of EGFR in biliary tract carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1082.

PI3K-mTOR pathway identified as a potential therapeutic target in biliary tract cancer using a newly established patient-derived cell panel assay.

Biliary tract carcinoma (BTC) is an extremely malignant tumor, but available treatment options are limited. Despite of needs for novel therapies, few BTC-related resources are currently available for evaluation of candidate drugs. To address this issue, we have recently established 13 cell lines from surgical specimens from Japanese BTC patients. In the present study, we evaluated four new molecular targeting agents using our BTC cell-based assay panel with 17 BTC cell lines. PI3K/mTOR dual inhibitor LY3023414 showed activity at submicromolar concentration ranges against 13 of the 17 cell lines tested, including the ones with gemcitabine insensitivity. In conclusion, we demonstrated that in vitro study with the BTC cell line panel would be an efficient approach to screen for novel therapeutic strategies. Although this is preliminary result and further investigations are required for confirmation, PI3K/mTOR inhibitor might be a potential target for BTC drug development.

Establishment of various biliary tract carcinoma cell lines and xenograft models for appropriate preclinical studies.

We recently reported several driver genes of biliary tract carcinoma (BTC) that are known to play important roles in oncogenesis and disease progression. Although the need for developing novel therapeutic strategies is increasing, there are very few BTC cell lines and xenograft models currently available for conducting preclinical studies. Using a total of 88 surgical BTC specimens and 536 immunodeficient mice, 28 xenograft models and 13 new BTC cell lines, including subtypes, were established. Some of our cell lines were found to be resistant to gemcitabine, which is currently the first choice of treatment, thereby allowing highly practical preclinical studies to be conducted. Using the aforementioned cell lines and xenograft models and a clinical pathological database of patients undergoing BTC resection, we can establish a preclinical study system and appropriate parameters for drug efficacy studies to explore new biomarkers for practical applications in the future studies.

Efficacy markers for cisplatin and S-1 in biliary tract carcinoma.

334 Background: Gemcitabine (GEM) + cisplatin (CDDP) combination chemotherapy is the first choice for advanced biliary tract carcinoma (BTC). In Japan, the GEM + S-1 therapy has shown promise for the treatment of BTC. Our aim was to investigate the cytotoxic effects of GEM, CDDP and S-1 on BTC cell lines and find an immunohistochemical marker for predicting the efficacy of chemotherapy. Methods: We evaluated the efficacy of GEM, CDDP, and S-1 by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of 17 BTC cell lines. Furthermore, we subdivided the cell lines into sensitive and insensitive groups on the basis of the sensitivity of each drug. Moreover, pairwise studies were conducted using the MTT assay, Bliss additivism model, and Combination Index for analysis. Immunohistochemical expression analysis of excision repair cross-complementation group 1 (ERCC1), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) were also performed in each cell line to compare the results of the cytotoxic effects of CDDP and S-1. Results: In the single-agent MTT assays, among the 17 tumor cell lines, 11 were sensitive to GEM, 13 to CDDP, and 11 to S-1. In 5 cell lines, <30% of the cells showed ERCC1 expression; these lines belonged to the CDDP-sensitive group. Conversely, the cell lines with >30% ERCC1-positive cells mostly belonged to the CDDP-insensitive group. CDDP IC50 correlated with the percentage of ERCC1-positive cells (P <.05). In contrast, none of the immunohistochemical markers could be used to select a cutoff level that would point to the sensitivity to S-1. In pairwise MTT assays (combination therapy), we demonstrated the synergy of GEM with CDDP in 9 tumor cell lines (53%) and the synergy of GEM with S-1 in 4 tumor cell lines(24%). Although 3 cell lines with the synergy of GEM with CDDP showed <30% ERCC1-positive cells, there was no correlation between them. Conclusions: ERCC1 expression in <30% of cells may help to identify CDDP-sensitive tumors; however, it is unknown whether expression of ERCC1 can help to predict efficacy of treatment with GEM + CDDP.

Anti-cancer effect and gene modulation of ET-743 in human biliary tract carcinoma preclinical models.

BackgroundStandard chemotherapy in unresectable biliary tract carcinoma (BTC) patients is based on gemcitabine combined with platinum derivatives. However, primary or acquired resistance is inevitable and no second-line chemotherapy is demonstrated to be effective. Thus, there is an urgent need to identify new alternative (chemo)therapy approaches.MethodsWe evaluated the mechanism of action of ET-743 in preclinical models of BTC. Six BTC cell lines (TFK-1, EGI-1, TGBC1, WITT, KMCH, HuH28), two primary cell cultures derived from BTC patients, the EGI-1 and a new established BTC patient-derived xenografts, were used as preclinical models to investigate the anti-tumor activity of ET-743 in vitro and in vivo. Gene expression profiling was also analyzed upon ET-743 treatment in in vivo models.ResultsWe found that ET-743 inhibited cell growth of BTC cell lines and primary cultures (IC50 ranging from 0.37 to 3.08 nM) preferentially inducing apoptosis and activation of the complex DNA damage-repair proteins (p-ATM, p-p53 and p-Histone H2A.x) in vitro. In EGI-1 and patient-derived xenografts, ET-743 induced tumor growth delay and reduction of vasculogenesis. In vivo ET-743 induced a deregulation of genes involved in cell adhesion, stress-related response, and in pathways involved in cholangiocarcinogenesis, such as the IL-6, Sonic Hedgehog and Wnt signaling pathways.ConclusionsThese results suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials in BTC patients resistant to standard chemotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-918) contains supplementary material, which is available to authorized users.

Overexpression and gene amplification of EGFR, HER2, and HER3 in biliary tract carcinomas, and the possibility for therapy with the HER2-targeting antibody pertuzumab

Pertuzumab is a humanized monoclonal antibody that binds to HER2 at an epitope that prevents HER2 from dimerizing with ligand-activated HER-family receptors. To assess the potential of pertuzumab as a new therapy, the expression status of HER family members was determined in biliary tract carcinoma (BTC), and the antitumor activity of pertuzumab was investigated by assessing the inhibition of BTC cell growth. The expression status of HER family members in 113 archival specimens of BTC was analyzed by using immunohistochemistry and fluorescence in situ hybridization. Using ten BTC cell lines, heregulin-alpha (HRG-α) stimulated cell proliferation and its inhibition by pertuzumab was tested in vitro. The phosphorylated HER family proteins and their respective downstream molecules were analyzed. In vivo antitumor activity of pertuzumab was evaluated in a xenograft model. Protein overexpression of HER2 and/or HER3 was observed in 23-34% of the specimens and gene amplification in 17-27%. Seven of the ten cell lines showed HER2 and/or HER3 protein overexpression and gene amplification, and HRG-α stimulated cell proliferation was observed in four of the ten cell lines. In a BTC cell line co-overexpressing HER2 and HER3, pertuzumab potently inhibited the HRG-α stimulated cell proliferation in a dose-dependent manner, and completely blocked the phosphorylation of HER3. Suppression of downstream pathway molecules including p-AKT was also observed. Pertuzumab inhibited the in vivo growth of subcutaneous tumors, and increased the number of apoptotic cancer cells. Pertuzumab exerts potent antitumor activity in BTC cells co-overexpressing HER2 and HER3. Pertuzumab provides a new therapeutic option against BTC.

Biliary tract carcinoma: clinical perspectives on molecular targeting strategies for therapeutic options

Biliary tract carcinoma (BTC) is a lethal malignancy. This lethality is essentially attributed to both slow carcinogenesis occurring under complex pathological circumstances and to the asymptomatic growth of BTC infiltrating the surrounding structures by varying routes. The disease is therefore usually detected at an advanced stage with a high frequency of distant organ metastasis. To date, conventional chemotherapy and radiation therapy have been notably ineffective against BTC. For an improved treatment outcome of BTC and prolonged survival, there is now a real and urgent need to focus on developing novel and potent therapeutic strategies aimed at exploiting select molecular targets associated with tumor proliferation, invasion, and/or metastasis that would impact in a significant way on clinical outcome. The outcomes of recent studies, by the analysis of BTC cells, BTC animal models, and clinical specimens of BTC patients, have revealed, in detail, the molecular mechanism of carcinogenesis and tumor progression of BTC, and these studies have exploited select molecular targets that could significantly impact the clinical outcome. In the near future, the development of new molecular targeting drugs with potent efficacy against BTC, and the performance of randomized clinical trials of these drugs are urgent and essential for the treatment of patients with BTC.

Influence of Preservation Solutions and Bile on a Human Bile Duct Epithelium Cell Line: An <i>In-Vitro</i> Study

Introduction: Bile duct complications are common after liver transplantation. The impact of preservation solution is unclear. Aim: We investigated the impact of different preservation solutions with and without diluted bile on a human biliary tract carcinoma cell line. Methods: The human biliary tree carcinoma cell line SK-ChA-1 was cultured with either medium or University of Wisconsin solution (UW), histidine-tryptophane-ketoglutarate (HTK) solution, Celsior or physiological saline for 6h, 10h or 12h at 6℃- 8℃ and metabolic activity was measured by a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-test immediately, after 12h or 24h. Cells were also incubated in combination with diluted porcine bile. Results: Immediately after cold storage of 6h HTK and UW decreased metabolic activity whereas Celsior increased metabolic activity after 10h or 12h of cold ischemia. After 12h or 24h no major differences were found any more. Incubation with bile in combination with HTK, Celsior or NaCl decreased metabolic activity, whereas UW abolished this effect. Conclusion: On a cellular level differences between preservation solutions were found, especially in combination with bile. Further studies are warranted to determine whether this results in clinically significant differences in biliary complications.

Ribonucleotide reductase subunit M1 assessed by quantitative double-fluorescence immunohistochemistry predicts the efficacy of gemcitabine in biliary tract carcinoma

Gemcitabine is a commonly used chemotherapeutic agent for advanced biliary tract carcinoma (BTC), although its efficacy is insufficient. Therefore, it is essential to establish new diagnostic methods, which can predict responders before the treatment. The aim of this study is to identify the most reliable chemoresistance marker to gemcitabine in BTC among the 4 molecules (hENT1, dCK, RRM1 and RRM2) involved in gemcitabine metabolism. The expression of 4 molecules were investigated in 5 BTC cell lines, and correlated with gemcitabine sensitivity. RRM1 protein was also assessed by quantitative double-fluorescence immunohistochemistry (qDFIHC) in 10 patients with unresectable or recurrent BTC who received gemcitabine-based chemotherapy. RRM1 and RRM2 protein strongly correlated with the IC(50) value for gemcitabine in BTC cell lines (R=0.935, 0.771, respectively). In addition, patients with low RRM1 were significantly more sensitive to gemcitabine (p=0.033), and their survival was significantly better than patients with high RRM1 (p=0.001). In conclusion, RRM1 particularly in protein level is a reliable marker for gemcitabine resistance in BTC. Furthermore, qDFIHC is a useful method for the assessment of RRM1 protein, in order to design a tailor-made chemotherapeutic regimen for BTC patients.

Novel photodynamic therapy against biliary tract carcinoma using mono‐l‐aspartyl chlorine e6: basic evaluation for its feasibility and efficacy

Recently, a second-generation photosensory agent for photodynamic therapy (PDT), mono-L: -aspartyl chlorine e6 (NPe6), which degrades rapidly in vivo, has been developed. We evaluated its feasibility and efficacy for treatment in biliary tract carcinoma. A transmittance of semiconductor laser light (664 nm), sensitivity of a human biliary tract carcinoma cell line, and disorder to normal tissue including Glissonian constructs and adjacent hepatocytes were investigated. The transmittance of the laser was 85-91% through yellow clear bile and that of the bile including 50 microg/ml NPe6 was 17-48%. The effective concentration of NPe6 which showed LD50 for a cell line was 12.5 microg/ml, and that of LD95 was 25 microg/ml. NPe6 in the supernatant reduced laser transmissiveness, but it had little influence on the antitumor effect in supernatant with or without NPe6. The NOZ cell-tumor volume was reduced significantly 14 days after irradiation in the PDT group (PDT 69.9 +/- 44.6 mm(3) vs control 296.3 +/- 239.9 mm(3) P < 0.05). No severe hepatic disorder including Glisson components was observed by the histological findings. NPe6 PDT was effective in carcinomas even in the presence of bile, and causes no serious complication for the liver and Glisson structure. Therefore, NPe6 PDT will be a useful candidate as a new therapy for biliary tract carcinomas.

Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment

The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome-wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine-treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine-treated recurrence cases, patients were categorized into "effective" and "non-effective" groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five "effective" cases and all four (100%) "non-effective" cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC.

Potent in vitro and in vivo antitumor activity of interleukin‐4‐conjugated Pseudomonas exotoxin against human biliary tract carcinoma

Targeting cytotoxins or immunotoxins to tumor cell surface receptors represents a new approach for the treatment of cancers. We tested the antitumor activity of a cytotoxin (IL-4-PE) composed of an interleukin-4 (IL-4) targeting moiety and a truncated form of Pseudomonas exotoxin A against human biliary tract carcinoma (BTC). Immunohistochemical analysis showed that cultured BTC cell lines and cancerous epithelia in BTC tissue (e.g., gallbladder carcinoma, extrahepatic cholangiocarcinoma, and intrahepatic cholangiocarcinoma) expressed receptors for IL-4 in situ at high densities. However, normal epithelial cells in gallbladder and bile duct tissues did not express these IL-4 receptors. Eight BTC cell lines expressed IL-4R on the cell surface as determined by radiolabeled ligand binding assays. When these cells were treated with IL-4-PE, significant cytotoxicity was observed as determined by the inhibition of protein synthesis. The concentration of agent causing 50% inhibition of protein synthesis (IC(50)) was found to be less than 10 ng/mL in 4 of the 8 BTC cell lines studied. The antitumor activity of IL-4-PE was assessed for human BTC cells implanted subcutaneously in immunodeficient mice. By intratumoral injection of IL-4-PE, complete disappearance of the established tumors was observed in 40% of animals. Intraperitoneal administration of IL-4-PE at tolerated doses to animals with peritoneally disseminated BTC exhibited significantly prolonged survival compared to untreated animals (>14 weeks vs. 5 weeks in treated and untreated mice, respectively). These results indicate that IL-4 receptor-targeted cytotoxin is a potent agent that may provide a new therapeutic option for BTC.

Frequent activation of mitogen-activated protein kinase relative to Akt in extrahepatic biliary tract cancer

Lack of effective adjuvant therapy against advanced extrahepatic biliary tract carcinoma (BTC) requires that new therapeutic methods, such as molecular targeted therapy, be developed. The mitogen-activated protein kinase (MAPK) and Akt signaling pathways, which activate cell proliferation and suppress apoptosis, respectively, may function as important targets for such therapies. The aim of this study was to examine the expression patterns of phosphorylated MAPK (p-MAPK) and phosphorylated Akt (p-Akt) proteins in BTC cell lines and clinical specimens. Expression of p-MAPK and p-Akt proteins in four human BTC cell lines and in frozen sections of 20 advanced extrahepatic BTC specimens was analyzed by Western blotting. Thirty formalin-fixed BTC specimens were immunohistochemically stained for p-MAPK and p-Akt using labeled streptavidin-biotin conjugates. Expression of p-MAPK was observed in three of four (75%) BTC cell lines, whereas no expression of p-Akt was observed. Twenty-three of 30 formalin-fixed specimens stained positive for p-MAPK (77%), whereas only 47% stained positively for p-Akt. Expression of p-MAPK relative to that of p-Akt was also seen more frequently in the frozen specimens. The results of this study suggest that MAPK is activated more frequently than Akt in extrahepatic biliary tract carcinoma.

Somatic mutations of EGFR signal transducers and expression of tumor suppressor PTEN in biliary tract carcinoma

4582 Background: Biliary tract carcinomas express EGFR and are potential candidates to EGFR target therapies. We recently described somatic mutations of EGFR that can enhance MAPK or Akt activation (Clin Cancer Res, 2006). Some of them are identical to those previously reported to confer sensitivity to some tyrosine kinase inhibitors (TKIs) like erlotinib or gefitinib in lung cancer. Here we report a molecular analysis of EGFR transducers potentially involved in TKI response. Methods: In 49 samples of biliary tract carcinoma we performed mutational analysis of exons from 18 to 21 of EGFR, exons 9 and 20 of phosphatidylinositol 3’-kinase (PI3K), exon 2 of K-Ras, exon 15 of B-Raf and exons from 5 to 8 of PTEN. Nuclear PTEN expression was analyzed by immunohistochemistry and the expression in cancer cells was compared to that of normal cholangiocites. Results: Mutations of EGFR have been detected in 7 out of 49 samples (14.3%). One of them was a new stop-codon mutation. Five hotspot mutations of PI3K (codon 545, 546, 1048 and 1059) were found in 4 cases (8.2%); 3 cases (6.1%) had single mutations in K-Ras and 4 (8.2%) had the V599E mutation in B-Raf. In some samples, mutations of multiple trasducers were present simultaneously. PI3K mutations were significantly more frequent in EGFR mutated samples compared to wild type (28% vs. 4.7% respectively, p&lt;0.05). A single F271L mutation of PTEN was observed (2%). We did not find loss of nuclear PTEN in biliary tract carcinoma cells; rather, a stronger labelling intensity (score 2+ or 3+) was present in biliary tract carcinoma compared to normal cholangiocites (score 1+). Moreover, the median percentage of PTEN labeled nuclei in tumor cells was 31.8%, but in samples with activating EGFR mutation was 60%, suggesting that a compensatory change in the level of the phosphatase might counteract the EGFR activition. Conclusions: These results corroborate our previous finding of EGFR pathway activation in EGFR mutated biliary tract carcinoma and suggest that an accurate analysis of the entire series of EGFR transducers may be done before planning treatments with TKIs. No significant financial relationships to disclose.

Re-expression of RASSF1A by 5-Aza-CdR induced demethylation of the promoter region in human biliary tract carcinoma cells

Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 micromol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1 kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Human equilibrative nucleoside transporter 1 is associated with the chemosensitivity of gemcitabine in human pancreatic adenocarcinoma and biliary tract carcinoma cells

Gemcitabine has been one of the most commonly used agents for pancreatic adenocarcinoma chemotherapy, but the determinants of the sensitivity of and resistance to this agent are not yet fully understood. In this study with pancreatic carcinoma and biliary tract carcinoma cell lines, we examined the gene expression levels of nucleotide transporters and others related to the metabolism of gemcitabine in the light of sensitivity to this agent. Quantitative RT-PCR demonstrated that one of the nucleotide transporter genes; human equilibrative nucleoside transporter 1 (hENT1) was associated with the sensitivity to gemcitabine as represented by IC50, while the other genes for nucleotide transporter and metabolism were not. We conclude that increased hENT1 expression is a most important determinant of gemcitabine sensitivity at least in an in vitro study.

Effect of antisense MBD1 gene eukaryotic expression plasmid on expression of MBD1 gene in human biliary tract carcinoma cells

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.

Skp2 overexpression is a p27Kip1‐independent predictor of poor prognosis in patients with biliary tract cancers

To better understand the pathogenesis of biliary tract carcinoma (BTC) and to increase the accuracy of predicting outcomes for patients with this disease, we performed molecular cytogenetic analyses of BTC cell lines and tumors to identify non-random amplification(s) and target gene(s) within the amplicons. Among several non random chromosomal aberrations detected in BTC cell lines by comparative genomic hybridization, gain/ampli-fication of DNA at 5p was the most frequently observed alteration. We assessed the copy number and expression status of the possible target gene SKP2 for 5p amplification in cell lines and 33 primary stage II or III tumors of BTC. SKP2 was amplified, and subsequently overexpressed in both cell lines and primary tumors of BTC. However, levels of Skp2 and p27Kip1 proteins were not correlated inversely. Heightened expression of Skp2 and reduced expression of p27Kip1 were both associated with a shorter disease-free and/or overall survival in univariate analyses. In multivariate regression analyses, Skp2 and p27Kip1 were independent predictive factors. Those results suggest that (a) overexpression of Skp2 through an amplification mechanism may contribute to the progression of BTC, (b) not only each molecule, but also the combination of Skp2 and p27Kip1, might be a useful predictor of the prognosis of BTC, and (c) molecular targets of Skp2 other than p27Kip1 may also be important factors in the pathogenesis of this disease.