BMC Cancer 2006, 6:165 http://www.biomedcentral.com/1471-2407/6/165 done with SPSS and the statistical package R [28]. The level of significance was set at p < 0.05. Results Expression of Ep-CAM in normal esophagus and esophageal SCC In a first step, we investigated the Ep-CAM expression in 10 normal, non-pathologic squamous epithelia of the esophagus. As previously reported [13], the non-patho- logic esophageal squamous epithelium was negative for Ep-CAM expression. In contrast four different levels of Ep- CAM expression (Figure 1), which were classified accord- ing to the Dako scoring system, were observed in the 70 esophageal SCC. We found 3+ Ep-CAM expression in 29/ 70 of the investigated tumors (41%). Eight of the 70 sam- ples were scored as 2+ (11%) and their membranous Ep- CAM staining was less intense, when compared to the 3+ samples. A weak Ep-CAM staining scored as 1+ was observed in 18/70 (26%) tumors and an absent expres- sion of Ep-CAM was detected in 15/70 tumors (21%). Neither in the complete patient collective (n = 70) nor in the group with clinical follow up (n = 53, Table 1 and 2, respectively) any statistical significant correlation between the level of Ep-CAM expression and the clinico-patholog- ical parameters pT-category, pN-category and grading were observed. It is important to note that a heterogene- ous Ep-CAM expression was seen at all expression levels and no correlation was observed between Ep-CAM heter- ogeneity within a tumor and its Ep-CAM expression score (Chi-square test, p = 0.134). Also, a specific staining pat- tern, e.g. intense staining at the invasive front, was not detected. Prognostic influence of Ep-CAM expression in esophageal SCC In the 53 patients with follow-up data and a median observation time of 15.0 months (range: 2 – 95 months) we found, that the general neo-expression of Ep-CAM (1+ - 3+) did not correlate with the patient's prognosis. The median cumulative disease-specific survival of the patients was 17.0 months for Ep-CAM 1+ - 3+ positive tumors and 24.0 months for Ep-CAM negative tumors (log rank, p = 0.3491; relapse-free survival: Ep-CAM 1+ - 3+ 16.0 months vs. Ep-CAM negative 43.0 months; log rank, p = 0.5993). Interestingly, tumors exhibiting a strong Ep-CAM expression (3+) conferred a significantly decreased median relapse-free survival interval. The median cumulative relapse-free survival interval of patients with Ep-CAM 3+ expression was 9.0 months, compared to 43.0 months for patients with 0 - 2+ Ep-CAM expression (log rank, p = 0.0001) (Figure 2A). The median disease-specific survival was 11.0 months for patients with Ep-CAM 3+ expressing tumors compared to > 24.0 months for those with 0-2+ Ep-CAM staining (log rank, p = 0.0003, Figure 2B). As expected, the pN-category was also of prognostic significance in our investigated series with a median disease-specific survival of 13.0 months for patients with positive nodal status compared to 75.0 months for patients without regional lymph node involvement (log rank, p = 0.0170). The prognostic value of the pT-category for disease-specific survival was not sta- tistically significant (pT1–2 vs. pT 3–4, 16 months vs. 28 months, respectively; log rank, p = 0.1178). Tumor grad- ing was also of no prognostic significance. For multivariate regression analysis we included Ep-CAM expression (Ep-CAM 3+ vs. Ep-CAM 0-2+), sex, age, pN, pT and grading. We found an independent prognostic influence for Ep-CAM 3+ on relapse-free survival and dis- ease-specific survival (p = 0.002 and p = 0.005, respec- tively) (Table 3). We also tested the possible interaction between the pN category and Ep-CAM expression within a multivariate model and confirmed the indepence of both, Ep-CAM and pN-category as independent prognos- tic factors (data not shown). Discussion Here we present the first comprehensive study of Ep-CAM expression in primary esophageal SCC and its impact on prognosis. In our study, almost 80% of the primary esophageal SCC showed de novo expression of Ep-CAM within the primary tumor, while normal squamous mucosa was negative for Ep-CAM expression. To quantify the level of Ep-CAM expression we used the evaluation cri- teria for the FDA approved HercepTest (Dako) for quanti- fication of the ERBB2 (HER2) gene product p185, the target for the therapeutic antibody trastuzumab (Hercep- tin). We used these guidelines because, comparable to Table 3: Multivariate survival analysis for relapse-free and tumor-related survival of patients with esophageal SCC after best model selection by BIC. Variable Relapse-free survival Lymph node metastasis Present vs. absent Ep-CAM expression 3+ vs. 0 - 2+ Tumor-related survival Lymph node metastasis Present vs. absent Ep-CAM expression 3+ vs. 0 - 2+ Relative risk of death (CI 95%) 3.068 (1.434 – 5.950) 5.392 (1.831 – 15.878) 2.924 (1.577 – 5.421) 3.762 (1.471 – 9.619) p-value < 0.001 < 0.001 < 0.001 < 0.001 Page 4 of 8 (page number not for citation purposes)