BMC Cancer 2006, 6:165 http://www.biomedcentral.com/1471-2407/6/165 Background Advances in surgical techniques over the last decade improved the outcome of patients with squamous cell car- cinomas (SCC) of the esophagus significantly. However, in comparison to other gastrointestinal malignancies, esophageal SCC belongs to the more aggressive tumors with 5-year survival rates averaging below 30 per cent [1,2]. From the survival data of patients receiving surgery with curative intention it is obvious, that at the time of ini- tial diagnosis in most patients unperceived tumor cell spread has occurred. The results of current multimodal adjuvant and neoadjuvant strategies for esophageal SCC to eliminate the minimal residual tumorload are still unsatisfactory and due to their unspecificity afflicted with significant side effects [3-5]. Therefore new adjuvant ther- apeutic concepts are urgently needed to eradicate effec- tively the minimal residual disease and to improve the post-operative prognosis of esophageal SCC patients. A promising basis for new systemic anti-cancer therapy represents the epithelial cellular adhesion molecule Ep- CAM, encoded by the 9-exon gene TACSTD1 [6,7] (Ep- CAM, EGP 40, GA733-2, 17-1A) that was recently re- mapped to chromosome 2p21 [8]. EpCAM is a 40 kD type I transmembrane glycoprotein with two epidermal growth factor like repeats in the external domain and a short intracellular domain consisting of two α-actin bind- ing sites for actin cytoskeleton linkage and functions as an intercellular adhesion molecule modulating cadherin- mediated adhesions and thereby adhesion strength [9- 12]. The physiologic expression of Ep-CAM in adult human tissues is strictly restricted to the basolateral cell membrane of glandular, pseudo-stratified and transi- tional epithelia, whereas normal squamous stratified epi- thelia are Ep-CAM negative [13]. Interestingly, de novo expression of Ep-CAM occurs during squamous cell car- cinogenesis of the oral cavity and of the lung[14]. The expression level increases during the progression from mild dysplasia to carcinoma [14]. Although the biological role of Ep-CAM in healthy tissues and in cancer is not understood conclusively, its overexpression is observed in several cancer types and has been associated with poor prognosis in breast cancer [15,16] and gallbladder cancer [17]. Of much interest, from the clinical point of view, is the possibility to use Ep-CAM as a target for immuno- therapy [18-21]. So far, very few data are available regard- ing Ep-CAM expression in esophageal cancer. Here we investigated the expression and prognostic impact of Ep- CAM in esophageal SCC to test the potential value of this molecule for antibody based adjuvant therapy in this aggressive cancer. Methods The ethics committee of the chamber of physicians of Hamburg approved this study. Informed consent was obtained from all patients before inclusion into the study. Tumor samples were collected from 70 patients with resectable esophageal carcinoma who had undergone rad- ical en bloc esophagectomy at the University Hospital Hamburg Eppendorf, Germany. Tumor stage and grade were classified by the routine histopathologic assessment according to the UICC (Union Internationale Contre le Cancer) Classification for Malignant Tumors [22,23] from pathologists unaware of the immunohistochemical find- ings. The survival analysis was calculated from 53 patients with R0 resection, where at least two months of prospec- tively evaluated clinical follow-up was available. Seven- teen patients were excluded from the survival analysis because of metastatic disease (n = 5), perioperative death (n = 5), non-tumor free resection margins (n = 5) and lost for follow-up (n = 2). The clinico-pathologic data are pre- sented in Table 1 (all patients) and in Table 2 (the 53 patients included in the survival analysis). Tissue preparation and immunohistochemistry The tumor tissue was snap-frozen in liquid nitrogen immediately after removal and stored at -80°C until use. The Ep-CAM antigen was detected with the monoclonal antibody Ber-EP4 (IgG1, Dako, Hamburg, Germany), which can be used on snap frozen material as described previously[24]. Briefly, 5 μm cryostat sections were cut from each tumor and were transferred onto glass slides pretreated with 3-triethoxysilyl-propylamin (Merck, Darmstadt, Germany). One section was stained with con- ventional hematoxylin an eosin (H & E) staining and the following section was stained using the alkaline phos- phatase-antialkaline phosphatase (APAAP) tech- nique[24]. Sections of normal colonic mucosa and MCF- 7 cells served as positive staining controls, and isotype- matched, irrelevant murine monoclonal antibodies served as negative controls (MOPC 21 for IgG1; Sigma, Deisenhofen, Germany). For the evaluation of the Ep- CAM staining we used standardized criteria to evaluate membranous staining specified by Dako for interpreta- tion of the HercepTest for p185HER2 expression. The Dako scoring system has a scale from 0 - 3+: 0, no staining or 10% or less of the tumor cells show any level of positive staining; 1+, a faint membrane staining is detected in more than 10% of the tumor cells or the cells are only stained in part of their membrane; 2+, weak to moderate staining of the entire membrane is observed in more than 10% of the tumor cells; 3+, strong staining of the entire membrane in more than 10% of the tumor cells. Hetero- geneous staining was defined as different staining inten- sity in more than 25% of Ep-CAM-positive tumor cells. The inter-observer reproducibility in our study was 93%. The slides with discrepant assessments were reevaluated, and a consensus was reached in all cases. Page 2 of 8 (page number not for citation purposes)