PROGNOSTIC SIGNIFICANCE OF Ep-CAM AND Her-2/neu OVEREXPRESSION IN INVASIVE BREAST CANCER Gilbert SPIZZO 1 , Peter OBRIST 2 , Christian ENSINGER 2 , Igor THEURL 2 , Martina D¨ UNSER 3 , Angela RAMONI 3 , Eberhard GUNSILIUS 1 , Gu ¨nther EIBL 4 , Gregor MIKUZ 2 and Gu ¨nther GASTL 1 * 1 Division of Hematology and Oncology, University of Innsbruck, Innsbruck, Austria 2 Institute of Pathology, University of Innsbruck, Innsbruck, Austria 3 Department of Surgery, University of Innsbruck, Innsbruck, Austria 4 Department of Biostatistics, University of Innsbruck, Innsbruck, Austria To assess the frequency and prognostic impact of Ep-CAM and Her-2/neu overexpression in patients with breast cancer and to determine its relationship with other prognostic markers, 205 breast cancer patients with a median follow-up of 10.8 years were enrolled in this retrospective study. Over- expression of Ep-CAM and Her-2/neu in tumor tissue sam- ples was assessed by immunohistochemistry. Tumors pre- senting a Her-2/neu 2 staining were additionally analyzed by FISH to exclude false positive results. Ep-CAM and Her- 2/neu overexpression was found in 35.6% and 19.5% of the tumor samples, respectively. Both Ep-CAM and Her-2/neu overexpression were predictive for poor disease-free (DFS) and disease-related overall survival (DROS). Concurrent Ep- CAM and Her-2/neu overexpression was present in 13.2% of tumor specimens and had an additive negative impact on DFS and DROS. This minority of patients had a median time to relapse of only 34 months, whereas the median time to relapse was not reached in the patient population without Her-2/neu and Ep-CAM overexpression. By multivariate analysis Ep-CAM overexpression proved to be an indicator of poor prognosis, independent of tumor size, histologic grade, hormone receptor expression and Her-2/neu overexpres- sion. In conclusion, overexpression of Ep-CAM and Her-2/neu complement each other as predictors for poor prognosis in patients with invasive breast cancer. Determination of these tumor markers should help in assigning breast cancer pa- tients to 1 of 3 distinct risk categories. © 2002 Wiley-Liss, Inc. Key words: breast; cancer; Ep-CAM; Her-2/neu The search for new prognostic factors in breast cancer reflects the need for a more adequate risk assessment to select the most appropriate treatment modalities for individual patients. Ep-CAM (also called 17-1A, ESA, EGP40, 323/A3) is a 40-kDa transmem- brane glycoprotein expressed on most human epithelial cells. 1 The encoding gene (GA733-2) has been sequenced and localized on chromosome 4. 2 Reportedly, Ep-CAM glycoprotein functions as a homotypic intercellular adhesion molecule. 3 In normal human mammary glands, Ep-CAM is mainly expressed in luminal epi- thelium. 4 Thus, tumors originating from luminal epithelium are likely to present Ep-CAM overexpression. We have recently re- ported that Ep-CAM overexpression, assessed by immunohisto- chemistry, in breast cancer predicts poor disease-free and overall survival, 5 corroborating findings from a previous study by Tandon et al. 6 using a western blotting technique. Among a variety of prognostic factors, Her-2/neu (also known as c-erbB-2) has recently attracted major scientific interest. Her- 2/neu oncogene amplification appears to be an important step in breast cancer tumorigenesis. The Her-2/neu gene encodes a trans- membrane 185-kD protein that shows homology to the epidermal growth factor family. 7,8 Her-2/neu gene amplification correlates with overexpression of the Her-2/neu protein. 9 Gene amplification and overexpression of Her-2/neu protein has been reported in 20% to 30% of breast carcinomas. 10 Her-2/neu overexpression was found to be associated with poor grade of differentiation, 11,12 hormone receptor negativity, 13 and poor survival. 14 –20 Patients with strong Her-2/neu expression seem to have a poorer response to hormonal agents such as tamoxifen 21 and a poorer response to non-anthracycline based chemotherapy. 22 Thus, Her-2/neu may become an important predictive marker in guiding therapeutic decisions. Recently, treatment with trastuzumab (Herceptin™), a humanized monoclonal antibody specific for Her-2/neu, either alone or in combination with chemotherapy has been shown to be efficacious in patients with Her-2/neu-overexpressing breast car- cinomas. 23–25 To screen for the Her-2/neu status it is recommended to use immunohistochemistry. Tumors with 2+ staining should additionally be assessed by FISH for evaluating gene amplifica- tion. In tumors with 3+ staining no additional diagnostic benefit can be expected by using FISH. 26 Ep-CAM can reportedly play a dual role in tumorigenesis. 27 Because Ep-CAM functions as an intercellular adhesion molecule, it has been claimed that overexpression could prevent shedding of cancer cells due to increased intercellular adhesion of tumor cells in primary lesions. This suggests that Ep-CAM overexpression might inhibit metastases. Only few studies support this hypothe- sis. 28,29 Ep-CAM overexpression was found to suppress cadherin- mediated cell adhesion. 30 It is known that E-cadherin functions as an invasion suppressor molecule. 31 Thus, Ep-CAM overexpression might promote tumor cell invasion and metastasis. Our results on the negative prognostic impact of Ep-CAM overexpression in breast cancer favor the latter hypothesis. 5 Studies in other epithe- lial cancers such as cervical cancer and hepatocellular carcinoma point to the same direction. 33,34 Furthermore, also Her-2/neu can disrupt the cadherin-catenin-mediated cell adhesion system, 35 sug- gesting a possible common molecular pathway of Ep-CAM and Her-2/neu. Because Her-2/neu is commonly used in the hospital setting and because specific antibody therapies are available for these 2 par- ticular markers we investigated in our study the relationship be- tween Her-2/neu and Ep-CAM overexpression in breast cancer and the prognostic impact of both tumor markers. PATIENTS AND METHODS A total number of 205 patients were included in this retrospec- tive study. This patient sample represents one-third of all cases with localized invasive breast cancer who were operated at the Department of Surgery, Innsbruck University Hospital, from 1980 –91. All cases for which paraffin-embedded tissue samples were still retrievable from the local pathology repository and for Grant sponsor: Tiroler Verein zur F¨ orderung der Krebsforschung. *Correspondence to: Division of Hematology and Oncology, Innsbruck University Hospital, Anichstr. 35, A-6020 Innsbruck, Austria. Fax: +43-5125045615. E-mail: Guenther.gastl@uibk.ac.at Received 18 July 2001; Revised 27 November 2001; Accepted 30 November 2001 Published online 8 February 2002 Int. J. Cancer: 98, 883– 888 (2002) © 2002 Wiley-Liss, Inc. DOI 10.1002/ijc.10270 Publication of the International Union Against Cancer