4072

EPCAM

epithelial cell adhesion molecule;EPCAM;epithelial cell adhesion molecule

adenocarcinoma-associated antigen|cell surface glycoprotein Trop-1|epithelial glycoprotein 314|human epithelial glycoprotein-2|major gastrointestinal tumor-associated protein GA733-2|membrane component, chromosome 4, surface marker (35kD glycoprotein)|tum

2p21

protein-coding

EpCAM (CD326) has diverse roles in cell adhesion and proliferation, and is known to be overexpressed in primary breast carcinomas (PBCs). While clinical and preclinical data suggest a role for EpCAM in metastases, the only prior study of EpCAM expression in breast cancer metastases suggested that EpCAM expression is decreased after first-line chemotherapy. This study evaluates EpCAM expression in metastatic breast carcinoma (MBC) versus matched PBC . Rapid autopsies were performed on 17 patients with widely metastatic breast cancer. Single patient tissue microarrays (TMAs) were constructed from archived PBC and post-mortem MBCs. In total, 169 spots from 17 PBCs and 895 spots from 195 MBCs were labeled for EpCAM by immunohistochemistry (IHC). expression was scored as intensity (1-3) multiplied by percent membrane labeling (0-100%) and was subclassified as low (0-100), moderate (101-200), or high (201-300) labeling. PBCs exhibited exclusively low-moderate EpCAM labeling. EpCAM labeling was present in all metastases and was significantly increased in MBCs of 14 of 17 patients (P value range <0.05 to <0.0001, t test). In the remaining three patients, EpCAM labeling was nonsignificantly increased in 1 and unchanged in 2. High EpCAM labeling was verified using a different antibody for IHC, as well as in a separate series of surgically resected metastases compared to unmatched surgically resected primary breast cancers. In conclusion, EpCAM is highly expressed in MBCs compared to matched PBCs, verifying that it is a promising therapeutic target.

Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention as a prognostic factor in breast cancer. We aimed to validate the influence of Ep-CAM RNA expression in untreated node-negative breast cancer. Ep-CAM RNA expression was evaluated utilizing microarray-based gene-expression profiling in 194 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. The prognostic significance of Ep-CAM RNA expression for disease-free survival (DFS), metastasis-free survival (MFS), and breast cancer-specific overall survival (OS) was evaluated in univariate and multivariate analysis adjusted for age, grading, pTstage, ER as well as PR receptor and HER-2 status. Additionally, Ep-CAM RNA expression was compared with immunohistochemistry (IHC) for Ep-CAM in 194 patients. The prognostic impact of Ep-CAM gene expression was validated in further 588 node-negative breast cancer patients. Levels of Ep-CAM RNA expression showed a significant correlation with IHC (P = 0.001) and predicted in univariate analysis DFS (P = 0.001, HR = 2.4), MFS (P = 0.003, HR = 2.5), and OS (P = 0.002, HR = 3.1) accurately. The prognostic influence of Ep-CAM RNA was significant also in multivariate analysis for DFS (P = 0.017, HR = 2.0), MFS (P = 0.049, HR = 1.9), and OS (P = 0.042, HR = 2.3), respectively. The association with MFS was confirmed in an independent validation cohort in univariate (P = 0.006, HR = 1.9) and multivariate (P = 0.035, HR = 1.7) analysis. Ep-CAM RNA correlated with the proliferation metagene (P < 0.001, R=0.425) Nevertheless, in multivariate analysis, Ep-CAM was associated with MFS independent from the proliferation metagene (P = 0.030, HR = 1.8). In conclusion, Ep-CAM RNA expression is associated with poor MFS in three cohorts of untreated node-negative breast cancer.

High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7-associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation.

OBJECTIVES: To investigate the correlation between the percentage of metastatic tumor present in lymph nodes resected from patients with squamous cell carcinoma of the head and neck (HNSCC) and level of expression of 3 marker genes: pemphigus vulgaris antigen (PVA), parathyroid hormone-related peptide (PTHrP), and tumor-associated calcium signal transducer 1 (TACSTD1). In addition, we investigated whether the level of expression of these 3 markers was associated with clinical outcomes for patients with HNSCC. STUDY DESIGN: Retrospective analysis of previously harvested patient samples. SETTING: The University of Pittsburgh. SUBJECTS AND METHODS: A total of 448 lymph nodes from 92 patients with HNSCC were evaluated for expression of the gene markers PVA, PTHrP, and TACSTD1 using real-time polymerase chain reaction. Confirmation of metastasis was determined by histologic examination. The expression level of these markers versus tumor percentage was analyzed. RESULTS: All 3 markers were studied independently and were associated with tumor percentage in metastatic lymph nodes. PVA had the strongest correlation, followed by PTHrP and then TACSTD1. PVA levels had a trend toward association with clinical outcome, specifically time to death caused by cancer, but this was confounded by tumor stage. CONCLUSION: All 3 tumor gene markers were associated with percentage of tumor cells in metastatic lymph nodes. PVA had the strongest correlation. PVA may add prognostic utility beyond pathologic staging, but this requires analysis of a larger cohort. Prospective studies of tumor volume in metastatic nodes should determine a lower limit threshold of molecular marker detection.

Current imaging technologies do not sufficiently detect micrometastasis and therefore do not allow adequate stratification of patients with hepatocellular carcinoma (HCC) for curative or systemic therapy. In HCC, presence of stem cell-like, epithelial cell adhesion molecule (EpCAM)-positive cells correlates with tumor aggressiveness and formation of metastasis. Therefore, we investigated the prognostic relevance of EpCAM-positive circulating tumor cells (CTCs) in patients with HCC. Blood from 78 patients (19 patients in the control cohort and 59 patients with HCC) was tested for CTCs with the CellSearch system. Correlation analysis to overall survival (OS), the Barcelona Clinic Liver cancer (BCLC) staging system, macroscopic and microscopic vascular invasion and alpha-fetoprotein (AFP) levels were performed. We detected >/=1 CTC in 18/59 HCC patients and in 1/19 patients with cirrhosis or benign hepatic tumor (p = 0.026). OS was significantly shorter (460 vs. 746 days) in the CTC-positive cohort (p = 0.017). Comparing BCLC stages, significant differences in CTC detection rates were also observed: BCLC stages A 1/9, B 6/31 and C 11/19 (p = 0.006). Ten of 18 patients with macroscopic and 10/16 patients with microscopic vascular invasion exhibited positive findings in CTC testing (p = 0.004 and p = 0.006). Furthermore, CTC results correlated to AFP (cutoff > 400 ng/mL) levels (p = 0.050). Our study demonstrates frequent presence of EpCAM-positive CTC in patients with intermediate or advanced HCC and its prognostic value for OS with possible implications for future treatment stratification.

The epithelial cell adhesion molecule (EpCAM) is involved in homophilic cell-cell adhesion in normal epithelia and is frequently overexpressed in primary and metastatic adenocarcinomas. It has been postulated that during detachment and dissemination of tumor cells, EpCAM may be down-regulated. Circulating tumor cells (CTC) may demonstrate this phenomenon as they have successfully escaped their local microenvironment and entered the circulation. EpCAM expression of CTC was compared to tumor cells in paraffin-embedded tissue arrays containing various benign diseases and carcinomas. EpCAM expression on CTC was determined by flow cytometry (FCM) and by immunohistochemistry (IHC) in paraffin-embedded tissue. To permit comparison of FCM results to those derived by IHC, EpCAM was quantified on cancer cell lines by FCM and then paraffin-embedded cell-blocks of these lines were used as staining guides for IHC analysis of tissue arrays. By IHC, 97% (384/397) of solid tissues analyzed had detectable EpCAM, with 72% of tissues showing antigen expression levels of > or =400,000 EpCAM molecules per cell. FCM analysis of CTC from 100 metastatic carcinoma patients with > or =2 CTC/90 microl blood showed EpCAM expression ranging from 9,900 to 246,000 (mean 49,700) antigens per cell. EpCAM expression was approximately 10-fold lower on CTC as compared to primary and metastatic tissues, suggesting that EpCAM expression is transient and dependent upon the local micro-environment. This supports the hypothesis that this adhesion molecule is down-regulated on carcinoma cells in the circulation.

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.

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.

BACKGROUND: Thyroid cancer is among the fastest growing malignancies; almost fifty-percent of these rapidly increasing incidence tumors are less than or equal to 1cm in size, termed papillary thyroid microcarcinoma (PTMC). The management of PTMC remains a controversy due to differing natural history of these patients. Epithelial cell adhesion molecule (EpCAM) is comprised of an extracellular domain (EpEx), a single transmembrane domain and an intracellular domain (Ep-ICD). Our group reported nuclear Ep-ICD correlated with poor prognosis in thyroid cancer (Ralhan et al., BMC cancer 2010,10:331). Here in, we hypothesized nuclear and cytoplasmic accumulation of Ep-ICD and loss of membranous EpEx may aid in distinguishing metastatic from non-metastatic PTMC, which is an important current clinical challenge. To test our hypothesis, Ep-ICD and EpEx expression levels were analyzed in PTMC and the staining was correlated with metastatic potential of these carcinomas. METHODS: Thirty-six PTMC patients (tumor size 0.5 - 1cm; metastatic 8 cases and non-metastatic 28 cases) who underwent total thyroidectomy were selected. The metastatic group consisted of patients who developed lymph node or distant metastasis at diagnosis or during follow up. The patients' tissues were stained for Ep-ICD and EpEx using domain specific antibodies by immunohistochemistry and evaluated. RESULTS: PTMC patients with metastasis had higher scores for nuclear and cytoplasmic Ep-ICD immunostaining than the patients without metastasis (1.96 +/- 0.86 vs. 1.22 +/- 0.45; p = 0.007 and 5.37 +/- 0.33 vs. 4.72 +/- 1.07; p = 0.016, respectively). Concomitantly, the former had lower scores for membrane EpEx than the non-metastatic group (4.64 +/- 1.08 vs. 5.64 +/- 1.51; p = 0.026). An index of aggressiveness, Ep-ICD subcellular localization index (ESLI), was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep - ICDnuc + Ep - ICDcyt + loss of membranous EpEx]. Notably, ESLI correlated significantly with lymph node metastasis in PTMC (p = 0.008). CONCLUSION: Nuclear and cytoplasmic Ep-ICD expression and loss of membranous EpEx were found to correlate positively with metastasis in PTMC patients. In addition, ESLI had the potential to identify metastatic behavior in PTMC which could serve as a valuable tool for solving a current dilemma in clinical practice.