EpCAM Overexpression Research Articles

Phase I Clinical Evaluation of Designed Ankyrin Repeat Protein [99mTc]Tc(CO)3-(HE)3-Ec1 for Visualization of EpCAM-Expressing Lung Cancer.

A high level of EpCAM overexpression in lung cancer makes this protein a promising target for targeted therapy. Radionuclide visualization of EpCAM expression would facilitate the selection of patients potentially benefiting from such treatment. Single-photon computed tomography (SPECT) using 99mTc-labeled engineered scaffold protein DARPin Ec1 has shown its effectiveness in imaging tumors with overexpression of EpCAM in preclinical studies, providing high contrast just a few hours after injection. This first-in-human study aimed to evaluate the safety and distribution of [99mTc]Tc(CO)3-(HE)3-Ec1 in patients with primary lung cancer. Twelve lung cancer patients were injected with 300.7 ± 103.2 MBq of [99mTc]Tc(CO)3-(HE)3-Ec1. Whole-body planar imaging (at 2, 4, 6 and 24 h after injection) and SPECT/CT of the lung (at 2, 4, and 6 h) were performed. The patients' vital signs and possible side effects were monitored up to 7 days after injection. The patients tolerated the injection of [99mTc]Tc(CO)3-(HE)3-Ec1 well, and their somatic condition remained normal during the entire follow-up period. There were no abnormalities in blood and urine tests after injection of [99mTc]Tc(CO)3-(HE)3-Ec1. The highest absorbed doses were in the kidneys, liver, pancreas, thyroid, gallbladder wall, and adrenals. There was also a relatively high accumulation of [99mTc]Tc(CO)3-(HE)3-Ec1 in the small and large intestines, pancreas and thyroid. According to the SPECT/CT, accumulation of [99mTc]Tc(CO)3-(HE)3-Ec1 in the lung tumor was found in all patients included in the study. Intensive accumulation of [99mTc]Tc(CO)3-(HE)3-Ec1 was also noted in regional metastases. [99mTc]Tc(CO)3-(HE)3-Ec1 can potentially be considered a diagnostic tracer for imaging EpCAM expression in lung cancer patients and other tumors with overexpression of EpCAM.

Overexpression of Epcam and CD133 Correlates with Poor Prognosis in Dual-phenotype Hepatocellular Carcinoma.

Background: Dual-phenotype hepatocellular carcinoma (DPHCC) is associated with high rate of post-operative recurrence and low rate of survival, which may reflect the post-operative persistence of cancer stem cells (CSCs). Here we explored the potential correlation between DPHCC and expression of CSCs markers.Methods: In this retrospective study, we included 19 patients with DPHCC and 61 patients with non-DPHCC treated in 2015 by liver resection. Paraffin-embedded tumor tissue specimens were analyzed using immunohistochemistry as well as immunofluorescence double-staining. Rates of recurrence-free survival and overall survival were compared between the two groups using the Kaplan-Meier method, and expression of the CSC markers CD133, CD90, and EpCAM were compared using real-time quantitative PCR and western blotting.Results: Overall survival rates were significantly lower for patients with DPHCC than patients with non-DPHCC at 1 year (78.9% vs 93.4%), 2 years (52.6% vs 72.1%), and 3 years (42.1% vs 67.2%) (P = 0.019). Multivariate Cox proportional hazard modeling identified CK19 positivity (P = 0.016) and multiple nodules (P = 0.023) as independent predictors of poor recurrence-free survival. Independent predictors of poor overall survival were CK19 positivity (P = 0.032), Barcelona Clinic Liver Cancer stage C (P = 0.025) and carbohydrate antigen 19-9 (CA19-9) >37 ng/ml (P = 0.016). Expression of CD133 and EpCAM mRNA and protein were significantly higher in DPHCC tissue than non-DPHCC tissue, while CD90 expression was similar between the groups.Conclusions: These results suggest that DPHCC is associated with significantly lower overall survival than non-DPHCC, and that the poor prognosis among DPHCC patients may be related to the presence of CSCs expressing CD133 and EpCAM.

The expression of EpCAM in extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare skin malignant tumor. The prognosis of EMPD with distant metastasis is poor, however an effective therapy has not yet established. Recently, EpCAM (epithelial cell adhesion molecule, CD326) has attracted attention as both prognostic marker and therapeutic target in several cancers. Besides, EpCAM is an important surface marker of circulating tumor cell (CTC) in the collection of CTC. Thus, the purpose of our study was to examine the expression levels of EpCAM and evaluate the correlation between its intensity of EpCAM and the clinical characteristics of EMPD. The expression of EpCAM in EMPD was examined using immunohistochemistry. Skin samples were obtained from 32 patients with EMPD. We found that almost all EMPD tissues (90.6%, 29/32) were positive for EpCAM. Furthermore, the staining intensity of EpCAM protein negatively correlated with the presence of distant metastasis. Overexpression of EpCAM in EMPD cells suggests that EpCAM may be a novel therapeutic target and the research of CTC may be newly developed in EMPD. Based on these findings, EpCAM may be a meaningful molecule in EMPD.

Abstract 4098: New strategy of drug response assessment using PDX organoid platform for non-resectable pancreatic cancer

Abstract To develop an efficient drug screening platform which overcomes the difference of drug response between initial screening and clinical trial stage is a pivotal issue for drug discovery. The patient-derived Xenograft (PDX) model has been reported as a screening system to reflect the microenvironment and heterogeneity of tumor. However, in pancreatic cancer that 80 % of patients is non-resectable, PDX is not be suitable for an initial screening model in terms of economic- and time cost of mouse-based amplification system as well as the lack of obtaining pancreatic tumor tissue from fine needle biopsy or percutaneous gun. To overcome this limitation, here we newly suggested organoids system, miniature organ culture on a dish, that are generated from tumor tissues of orthotopic PDX model, which has the advantages of reflection of each patient's characteristics as well as amplification of limited tumor tissue. Besides, it is possible to screen of drug responsibility with a little number of cells. 12 organoids derived from PDX using needle or gun biopsy tumor tissues showed EpCAM overexpression and each unique morphological phenotype. Moreover, from drug responsibility test, H #43 and H #44, an organoids derived from a gemcitabine-sensitive patients, were highly responsible to gemcitabine, whereas the organoids from gemcitabine-resistant patients, G #20 and H #19 showed a strong resistance to gemcitabine as measuring the IC50 value. In addition, combined treatment with gemcitabine and abraxane to the G #13 model which has no clinical information of drug response due to early death, it inhibited organoid formation significantly, showing a combination index below 1, which was proved through in vivo (PDX) validation. Taken together, the PDX-Organoid system might be able to reflect primary tumor characteristics as well as to overcome the quantitative limitations of the specimen and time cost, and thereby it is possible to predict drug response early in vitro, making it very efficient as an anti-cancer drug development platform for pancreatic cancer. Citation Format: A-Ra Jeon, Sun Il Choi, Sang-Jae Park, Sung-Sik Han, Sun-Young Kong, Min Kyeong Kim, Yu-sun Lee, Jieun Im, Min Kyeong Lee, Sang Hyun Park, Joon-Ki Kim, Kyong-Ah Yoon, Young-Hwan Koh, Ju Hee Lee, Woo Jin Lee, Sang Myung Woo, Yun-Hee Kim. New strategy of drug response assessment using PDX organoid platform for non-resectable pancreatic cancer [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 4098.

Oncogene and therapeutic target analyses in atypical fibroxanthomas and pleomorphic dermal sarcomas.

BackgroundUntil now, almost nothing is known about the tumorigenesis of atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS). Our hypothesis is that AFX is the non-infiltrating precursor lesion of PDS.Materials and MethodsWe performed the world-wide most comprehensive immunohistochemical and mutational analysis in well-defined AFX (n=5) and PDS (n=5).ResultsIn NGS-based mutation analyses of selected regions by a 17 hotspot gene panel of 102 amplicons we could detect TP53 mutations in all PDS as well as in the only analyzed AFX and PDS of the same patient. Besides, we detected mutations in the CDKN2A, HRAS, KNSTRN and PIK3CA genes.Performing immunohistochemistry for CTNNB1, KIT, CDK4, c-MYC, CTLA-4, CCND1, EGFR, EPCAM, ERBB2, IMP3, INI-1, MKI67, MDM2, MET, p40, TP53, PD-L1 and SOX2 overexpression of TP53, CCND1 and CDK4 was seen in AFX as well as in PDS. IMP3 was upregulated in 2 AFX (weak staining) and 4 PDS (strong staining).FISH analyses for the genes FGFR1, FGFR2 and FGFR3 revealed negative results in all tumors.ConclusionsUV-induced TP53 mutations as well as CCND1/CDK4 changes seem to play essential roles in tumorigenesis of PDS. Furthermore, we found some more interesting mutated genes in other oncogene pathways (activating mutations of HRAS and PIK3CA). All AFX and PDS investigated immunohistochemically presented with similar oncogene expression profiles (TP53, CCND1, CDK4 overexpression) and the single case with an AFX and PDS showed complete identical TP53 and PIK3CA mutation profiles in both tumors. This reinforces our hypothesis that AFX is the non-infiltrating precursor lesion of PDS.

MMTV/LTR Promoter-Driven Transgenic Expression of EpCAM Leads to the Development of Large Pancreatic Islets.

Our previous work demonstrated an important role of EpCAM in the regulation of pancreatic cell adhesion, growth and differentiation. Here we investigated the consequences of human EpCAM (hEpCAM) overexpression under the control of the MMTV-LTR promoter, known to drive robust gene expression in a number of ductal epithelia, including the pancreas. In this animal model (MMTV-hEpCAM) we uncovered a striking pancreatic phenotype exhibiting a 12-fold increase in the islet cell mass, with normal expression patterns of insulin and the transcription factor PDX-1. Intriguingly, these large islet clusters revealed an altered architectural organization of α- and δ-cells that appeared interspersed with β-cells in the islet cores. This suggests an effect of the hEpCAM transgene on the function of other cell adhesion molecules that we have previously shown to regulate islet cell type segregation. Consistent with this finding, we show that the pancreatic epithelium in MMTV-hEpCAM transgenic mice exhibits a redistribution of β-catenin, a known regulator of E-cadherin-mediated adhesions. Collectively, these results provide an important in vivo validation of hEpCAM signaling properties in normal epithelia and offer unique opportunities to further explore the function of this glycoprotein in select pancreatic cell lineages to elicit islet cell expansion, and/or regeneration in diabetes.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.

Alteration of cell-cell and cell-matrix adhesion in urothelial cells: an oncogenic mechanism for mutant FGFR3.

Activating mutations of FGFR3 are a common and early event in bladder cancer. Ectopic expression of mutant FGFR3 in normal urothelial cells has both pro-proliferative and antiapoptotic effects at confluence, suggesting that mutant cells are insensitive to cell-cell contact inhibition. Herein, detailed analysis revealed that these cells have reduced cell-cell adhesion, with large intercellular spaces observable at confluence, and diminished cell-substrate adhesion to collagen IV, collagen I, and fibronectin. These phenotypic alterations are accompanied by changes in the expression of genes involved in cell adhesion and extracellular matrix remodeling. Silencing of endogenous mutant FGFR3 in bladder cancer cells induced converse changes in transcript levels of CDH16, PLAU, MMP10, EPCAM, TNC, and HAS3, confirming them as downstream gene targets of mutant FGFR3. Overexpression of EPCAM, HAS3, and MMP10 transcripts was found in a large fraction of primary bladder tumors analyzed, supporting their key role in bladder tumorigenesis in vivo. However, no correlation was found between their protein and/or mRNA expression and FGFR3 mutation status in tumor specimens, indicating that these genes may be targeted by several converging oncogenic pathways. Overall, these results indicate that mutant FGFR3 favors the development and progression of premalignant bladder lesions by altering key genes regulating the cell-cell and cell-matrix adhesive properties of urothelial cells. The ability of mutant FGFR3 to drive transcriptional expression profiles involved in tumor cell adhesion suggests a mechanism for expansion of premalignant urothelial lesions.

Clinicopathologic Implications of EpCAM and Sox2 Expression in Breast Cancer

BackgroundThe purpose of this study was to investigate the clinicopathologic significance of EpCAM and Sox2 expression in breast cancer and to study their correlation during breast cancer progression. Patients and MethodsEpCAm and Sox2 expression were assessed using immunohistochemistry in ductal carcinoma insitu (DCIS), invasive breast cancer (IBC) and matched lymph node metastasis (LNM), if present. ResultsEpCAM overexpression was found in 63.2% of DCIS, 72.2% of IBC and 74.4% of LNM. In IBC cases, EpCAM overexpression was associated with high grade (P < .001), large tumor size (P = .051), poor Nottingham Prognostic Index (NPI) (P = .006), histological tumor types (P = .044) and the triple negative phenotype (P = .008). LNM frequently reflected the expression phenotype of the matched primary tumors with no significant differences between LNM and their primary tumors (P = .564). Sox2 expression was detected in 47.4%, 33.3% and 54.7% of DCIS, IBC and LNM respectively. In DCIS group, Sox2 expression was significantly associated with comedo type (P = .037), negative ER (P = .012) and PR (P = .037) and the triple negative phenotype (P = .006). In IBC cases, Sox2 expression showed significant associations with high grade (P = .045), nodal spread (P = .037), poor NPI (P = .018) and the triple negative phenotype (P < .001). LNM showed significantly higher Sox2 expression rates than primary tumors (P < .001). Significant positive associations between EpCAM overexpression and Sox2 positivity in DCIS (P = .027), IBC (P = .001) and LNM (P < .001) were found. ConclusionThis study emphasized the potential role of EpCAM and Sox2 in breast carcinogenesis and revealed their involvement during breast cancer progression and LN metastases.

Cysteine-Poor Region-Specific EpCAM Monoclonal Antibody Recognizing Native Tumor Cells with High Sensitivity

EpCAM is a ∼40 kDa transmembrane glycoprotein. EpCAM overexpression is a popular trait of almost all carcinomas and is considered as a targeted cancer immunotherapy as well as a practical marker for circulating tumor cells (CTC). Its extracellular part (EpEx) consists of an N-terminal EGF-like (EGF) domain, a TY-like (TY) domain, and an uncharacterized cysteine-poor (CP) region. Most commercially available murine monoclonal antibodies (MAbs) to EpCAM, such as HEA 125 and VU-1D9, bind to the small EGF domain. In a previous study, we introduced iCeap (intact CTC enumeration and analysis procedure), keeping cellular integrity during the whole process. Unlike the CellSearch(®) CTC Test, iCeap enables downstream molecular analysis from detected CTC. Use of two EpCAM MAbs, one for immunomagnetic enrichment of rare CTC from blood samples and the other for labeling, is a concept of iCeap while an ideal MAb pair has not been found. In order to obtain a better MAb that recognizes a part of EpEx as different from EGF domain, we established a mouse hybridoma clone producing a new EpCAM MAb, KIJY2. Fluorophore-conjugated KIJY2 and HEA 125-FITC can concomitantly stain the tumor cell line LNCaP within indistinguishable cellular compartments (i.e., the cell surface). Epitope mapping reveals that KIJY2 binds to the CP region. The epitope for KIJY2 is sensitive to paraformaldehyde fixation, but native cells including MCF-7 (EpCAM high-expressing cell line) and PC-3 (EpCAM low and heterogeneously expressing cell line) are detected by KIJY2. In particular, KIJY2 detects all PC-3 cells regardless of their EpCAM expression levels. Therefore, KIJY2 and an EGF domain-directed MAb are a promising pair to form the EpCAM sandwich in iCeap. We demonstrate that KIJY2 incorporated into iCeap yielded favorable results in spike-in experiments of MCF-7 and PC-3.

Molecular characterization of circulating tumor cells in patients with ovarian cancer improves their prognostic significance — A study of the OVCAD consortium

ObjectiveThe study aims at identifying novel markers for circulating tumor cells (CTCs) in patients with epithelial ovarian cancer (EOC), and at evaluating their impact on outcome. MethodsMicroarray analysis comparing matched EOC tissues and peripheral blood leucocytes (N=35) was performed to identify novel CTC markers. Gene expression of these novel markers and of EpCAM was analyzed using RT-qPCR in blood samples taken from healthy females (N=39) and from EOC patients (N=216) before primary treatment and six months after adjuvant chemotherapy. All samples were enriched by density gradient centrifugation. CTC positivity was defined by over-expression of at least one gene as compared to the healthy control group. ResultsCTC were detected in 24.5% of the baseline and 20.4% of the follow-up samples, of which two thirds were identified by overexpression of the cyclophilin C gene (PPIC), and just a few by EpCAM overexpression. The presence of CTCs at baseline correlated with the presence of ascites, sub-optimal debulking, and elevated CA-125 and HE-4 levels, whereas CTC during follow-up occurred more often in older and platinum resistant patients. PPIC positive CTCs during follow-up were significantly more often detected in the platinum resistant than in the platinum sensitive patient group, and indicated poor outcome independent from classical prognostic parameters. ConclusionsMolecular characterization of CTC is superior to a mere CTC enumeration or even be the rationale for CTC diagnostics at all. Ultimately CTC diagnostics may lead to more personalized treatment of EOC, especially in the recurrent situation.

Abstract 5333: EpCAM overexpression in metastasizing gastric carcinomas

Abstract EpCAM, a homophilic Ca2+-independent adhesion molecule, is expressed in various epithelial tissues. Especially proliferating tissues or neoplasias show an increasing expression of EpCAM. Since it is found in large quantities in carcinomas, it seemed to be useful as novel therapeutic target for monoclonal and bispecific antibodies raised against the protein. Due to lack of data in gastric carcinomas, we investigated these tumors and their corresponding metastases. Therefore we evaluated the immunohistochemical reactivity of 104 gastric cancers, and their lymph node as well as distant metastases, using the mouse monoclonal antibody ESA by use of a previously defined scoring system for overexpression. Most of the tumour cells showed distinct membraneous reactivity, while normal gastric epithelium as internal control was EpCAM negative. 104 cases presented with an overexpression in 65,39%, without significant correlation to tumour stage or grade (p=0,339), no significant difference to histological growth patterns (according to Lauren) was found (p=0,186). 46,15 % of 26 mucinous carcinomas showed an overexpression, without mucinous parts they had an overexpression of 68 %, resulting in highly significant differences (p = 0.039 - two tailed) in these two groups. 54 primary tumors with metastases were evaluated. 62.96 % of these primary tumours had an EpCAM overexpression. 52 cases with lymph node metastases revealed a positive correlation with high significance (Pearson, p &amp;lt; 0.05). The paired-samples T-test no significance was found (p = 0.283 - two tailed). Comparing primary tumors with distant metastases a proportional relationship between the values was found (Pearson). The t-test brought no significant differences (p = 0.120). Between the lymph node metastases and distant metastases a positive correlation could be seen (Pearson, p = 0.192), without significance in the t-test (p = 0.121). Our study shows an EpCAM overexpression already in early stages of gastric cancers and a significant correlation between the primary tumors and metastases. So it seems to be possible to conclude from the EpCAM evaluation in available tissue of the primum to its metastases, facilitating clinical use of EpCAM-based immunotherapies and provide higher efficiency in the fight against all kinds of metastasizing gastric carcinomas. Recently designed new bispecific monoclonal antibodies are exerting improved therapeutic effects through antibody-dependent cellular cytotoxicity or complement. Also promising results were described for experimental gene therapy using EpCAM-targeted adenoviral vectors in human gastric and oesophageal carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5333. doi:1538-7445.AM2012-5333

P3-17-10: EpCAM as a Target for Chemical Antibodies in Metastatic Breast Cancer.

Abstract EpCAM is a 40kDa glycoprotein that functions as an adhesion molecule in benign epithelium. However, it is over-expressed in nearly all carcinomas, including breast carcinomas, and has been implicated in the pathogenesis and progression of cancer. In primary breast carcinoma, its over-expression correlates with a diminished overall survival in patients with node-positive disease. Indeed, recent evidence has shown that EpCAM is expressed at a higher level in metastases than in matched primary breast carcinoma. This has led to the suggestion that EpCAM over-expression is associated with aggressive behaviour, and that it could be considered a promising therapeutic target for breast cancer. Several immunotherapy trials are ongoing, though there are limitations to their delivery and distribution, suggesting a smaller and more effective EpCAM targeting molecule is required. Aptamers, also known as chemical antibodies, are short-stranded DNA or RNA molecules and are selected via the systematic evolution of ligands by exponential enrichment (SELEX). They are 10 to 20 times smaller than antibodies and exhibit superb tissue penetration, thereby indicating their suitability in cancer targeting. In addition, aptamers are more stable, non-immunogenic and non-toxic. Following 12 rounds of SELEX using EpCAM protein as a target, we generated several EpCAM specific RNA aptamers. These were investigated using flow cytometry and confocal microscopy for their ability to bind to three breast cancer cell lines, MCF-7, T47D and MDA-MB-231. Our 19-nt aptamer showed specific binding to all three cell lines. In addition, it also showed superior penetration of breast cancer 3-D spheroid tumor models, as compared to a control aptamer and negative cell lines. This aptamer has also shown efficient internalisation via receptor-mediated endocytosis, suggesting it to be a suitable targeting moiety for drug delivery to both primary and metastatic tumors. The usefulness of this aptamer in clinical applications is being actively investigated. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-10.

Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer.

Background:The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma.Methods:Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2′-deoxycytidine (5-AZAC) was used to induce EpCAM expression.Results:Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-κB probe.Conclusion:This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression.

EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis

AimsEpithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more...

Effects of EpCAM overexpression on human breast cancer cell lines

BackgroundRecently, EpCAM has attracted major interest as a target for antibody- and vaccine-based cancer immunotherapies. In breast cancer, the EpCAM antigen is overexpressed in 30-40% of all cases and this increased expression correlates with poor prognosis. The use of EpCAM-specific monoclonal antibodies is a promising treatment approach in these patients.MethodsIn order to explore molecular changes following EpCAM overexpression, we investigated changes of the transcriptome upon EpCAM gene expression in commercially available human breast cancer cells lines Hs578T and MDA-MB-231. To assess cell proliferation, a tetrazolium salt based assay was performed. A TCF/LEF Reporter Kit was used to measure the transcriptional activity of the Wnt/β-catenin pathway. To evaluate the accumulation of β-catenin in the nucleus, a subcellular fractionation assay was performed.ResultsFor the first time we could show that expression profiling data of EpCAM transfected cell lines Hs578TEpCAM and MDA-MB-231EpCAM indicate an association of EpCAM overexpression with the downregulation of the Wnt signaling inhibitors SFRP1 and TCF7L2. Confirmation of increased Wnt signaling was provided by a TCF/LEF reporter kit and by the finding of the nuclear accumulation of ß-catenin for MDA-MB-231EpCAM but not Hs578TEpCAM cells. In Hs578T cells, an increase of proliferation and chemosensitivity to Docetaxel was associated with EpCAM overexpression.ConclusionsThese data show a cell type dependent modification of Wnt signaling components after EpCAM overexpression in breast cancer cell lines, which results in marginal functional changes. Further investigations on the interaction of EpCAM with SFRP1 and TCF7L2 and on additional factors, which may be causal for changes upon EpCAM overexpression, will help to characterize unique molecular properties of EpCAM-positive breast cancer cells.

Abstract 4251: Murine hepatocellular carcinoma stem cells express pluri-potency-associated transcription factors and are sensitive to immune-mediated apoptosis

Abstract Worldwide the incidence of hepatocellular carcinoma (HCC) resulting from hepatitis C infection and chronic inflammation is increasing. Poor prognosis HCC is characterized by over-expression of Ep-CAM and alpha-fetoprotein (AFP) which define a population of tumor-initiating cells with stem/progenitor features. Current evidence supports the view that cancer stem cells (CSCs) constitute the primary pathogenic component of both metastases and minimal residual disease, and that elimination of CSCs is required for cure. Typically cell surface markers have been used to identify CSCs, however cell surface phenotype alone does not identify CSC in all tumors from the same tissue or in all patients. Therefore there is a need to define and test more reliable markers of CSCs. Oct-4, Sox-2 and Nanog transcription factors are key regulatory factors in maintenance of pluripotency and have been demonstrated to be robust intracellular biomarkers for tissue and cancer stem cells. A GFP-based reporter driven by a promoter responsive to Nanog or Oct-4 was used to mark pluripotent tumor cells from in vitro cultured, cancer cell lines (breast cancer- 4T1.2), and from autochthonous hepatocellular carcinoma induced by hydrodynamics-based gene transfer of constituitively active Akt and β-Catenin. This tumorigenesis model allows tumor initiation by delivery of clinically relevant oncogenes whose sporadic distribution reflects spontaneous cancer. The molecular profile of primary Akt/β-Cat-induced murine HCC recapitulates that observed in poor prognosis Ep-CAM+/AFP+ human HCC. Further, in vitro functional analyses suggest that Ep-CAM and CD133 (surface markers) and Nanog and Oct-4 (transcription factors required for maintenance and regulation of pluripotency in undifferentiated cells) are CSC markers in this model of HCC. Directly ex vivo, murine HCC stem cells express abundant CD1d and MHC class II in addition to TNF-R2, lymphotoxin-β receptor (LTβR) and DR5. The functional significance of CD1d, MHC class II, TNF-R2, LTβR and DR5 expression will be assessed in future studies using in vitro apoptosis and cytotoxicity assays and transplantation at orthotypic sites prior to adoptive transfer of tumor-specific T cells. Studies of freshly isolated CSCs have demonstrated that a variety of mechanisms combine to render CSC resistant to conventional chemo- and radiotherapies. This study has combined in vitro and in vivo approaches to address whether CSC can be rendered sensitive to T cell-mediated apoptosis, and will provide valuable proof-of-principle data to the merits of CSCs as targets for curative anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4251.

Increased EpCAM expression in malignant insulinoma: potential clinical implications

EpCAM (CD326) is overexpressed in progenitor cells of endocrine pancreatic islands of Langerhans during fetal development and was suggested to act as a morphoregulatory molecule in pancreatic island ontogeny. We tested whether EpCAM overexpression is reactivated in insulinomas, endocrine tumors arising in the pancreas. We used monoclonal anti-EpCAM antibody Ber-Ep4 for immunohistochemistry on formalin-fixed and paraffin-embedded tumor material. We analyzed 53 insulinomas: 40 benign (disease stage<IIa) and 13 malignant tumors (disease stage IIIb/IV). Disease stage disposition followed new TNM classification of the European Neuroendocrine Tumor Society (ENETS) for foregut neuroendocrine tumors (2006). Additionally, ten insulinoma metastases were analyzed. Clinical follow-up was available for overall survival analysis from 49 patients. The EpCAM expression of the islands of Langerhans was classified as 2+ in healthy pancreatic tissue. In 38% of the benign insulinomas (disease stage<IIa), we found strong (3+) EpCAM expression. In contrast, malignant insulinomas (disease stage IIIb/IV) and their metastases exhibited a strong (3+) EpCAM expression with 78 and 80% respectively, significantly more frequent (P<0.01). The malignant tissue was characterized by a significantly lower number of unstained cells and significantly higher number of 3+ stained cells. Quantitative PCR for EpCAM mRNA validated strong EpCAM expression in malignant insulinoma. Kaplan-Meier curves indicated survival disadvantage for EpCAM 3+ insulinomas, but this was not statistically significant (log-rank test). This first EpCAM expression study in benign/malignant insulinomas indicates that strong EpCAM expression could help to identify patients at risk for malignant disease and might be used as a therapeutic target for antibody-based therapies in patients with metastatic insulinoma.

Overexpression of Epithelial Cell Adhesion Molecule in Primary, Metastatic, and Recurrent/Chemotherapy-Resistant Epithelial Ovarian Cancer

To evaluate the potential of epithelial cell adhesion molecule (Ep-CAM/TROP-1)-specific immunotherapy against epithelial ovarian carcinomas (EOCs), we have analyzed the expression of Ep-CAM at RNA and protein level in patients harboring primary, metastatic, and chemotherapy-resistant/recurrent EOC. Epithelial cell adhesion molecule expression was evaluated by real-time polymerase chain reaction and immunohistochemistry in 168 fresh-frozen biopsies and paraffin-embedded tissues. In addition, Ep-CAM surface expression was evaluated by flow cytometry in several freshly established ovarian carcinoma cell lines derived from patients harboring tumors resistant to chemotherapy in vivo as well as in vitro. Epithelial cell adhesion molecule transcript was found significantly overexpressed in primary, metastatic, and recurrent EOC when compared with normal human ovarian surface epithelium cell lines and fresh-frozen normal ovarian tissue (P < 0.001). Similarly, by immunohistochemistry, Ep-CAM protein expression was found significantly higher in primary, metastatic, and recurrent EOC when compared with normal ovarian tissues. Of interest, metastatic/recurrent tumors were found to express significantly higher levels of Ep-CAM protein when compared with primary ovarian carcinomas (P < 0.001). Finally, a high surface expression of Ep-CAM was found in 100% (5/5) of the chemotherapy-resistant ovarian carcinoma cell lines studied by flow cytometry. These results demonstrate high Ep-CAM overexpression in ovarian carcinoma, especially in metastatic and recurrent/chemotherapy-resistant ovarian disease. The lack of Ep-CAM expression on the chelomic epithelium in the peritoneal cavity, combined with the recent development of fully human monoclonal antibodies against this surface molecule, suggest Ep-CAM as a promising target for antibody-mediated therapies in ovarian carcinoma patients harboring tumors refractory to standard treatment modalities.

Research advances of EpCAM gene and tumor

EpCAM gene plays a significant role in modulation of proliferation,differentiation,and migration of epithelial cells.Researches show that EpCAM contributes to tumorigenesis by promoting the cell proliferation and facilitating the immune escape.EpCAM is widely expressed in most epithelial carcinomas,and EpCAM over-expression is related to poor prognosis.It has been recently demonstrated that new technologies and agents based on EpCAM-antibodies can detect tumour cells accurately and promptly,and hence kill them specifically.Therefore,EpCAM could be used as a new marker in the diagnosis,treatment and prognosis of carcinoma. Key words: Neolplasms; Prognosis; EpCAM gene