Retrieval of tissue and clinical data was performed according to the regulations of the local institutional review board and data safety laws. The grade of the tumours was sellekchem obtained by a review of each case by a specialised pathologist. A total of 1407 colon cancers (1261 adenocarcinomas NOS, five medullary carcinomas, 119 mucinous carcinomas, five signet ring carcinomas, four other types), 559 stomach, 1527 lung (367 adenocarcinomas (AC), 13 adenosquamous carcinomas, 82 bronchioloalveolar carcinomas, 258 large cell carcinomas, 63 neuroendocrine carcinomas, 744 squamous cell carcinomas (SCC)) and 553 prostate carcinomas were then arrayed as described before (Bubendorf et al, 2001). Briefly, tissue cylinders with a diameter of 0.6mm were punched from representative tumour areas of each donor tissue block and brought into a recipient paraffin block.

Multiple 4��m sections of the resulting tissue microarray block were cut and mounted to an adhesive-coated slide system. Clinical data All relevant patient data were anonymised. Sex, age and stage according to the WHO/UICC 1997 were recorded in subsets of gastric, colon, lung and prostate cancers. In prostate cancers, the Gleason score was available. Grade was also known in colon and lung cancers. Additionally, the survival time was recorded in tumours from the colon, lung and prostate, but not stomach. Overall survival (OS) was calculated from the date of diagnosis until death from any cause or date of last contact for living patients. The cause of death was recorded in a subset of patients to calculate the tumour-specific survival.

Immunohistochemistry Standard indirect staining procedures were used for immunohistochemistry (ABC-Elite-Kit, Vector Laboratories, Burlingame, CA, USA). After heat-induced pretreatment (water bath, 30min, 99��C in target retrieval solution buffer (DAKO code S1699, DAKO, Glostrup, Denmark)) for antigen retrieval, a mouse monoclonal anti-Ep-CAM-antibody (ESA, clone VU-1D9, Novocastra, Newcastle upon Tyne, UK) was applied for 2h at a dilution of 1:50 at 37��C. The slides were then incubated with the secondary, biotinylated antibody. Osmium-enhanced diaminobenzidine was used as the chromogen. Counterstaining was carried out with Harris’ haematoxylin. Only fresh cut slides were stained simultaneously to minimise the influence of slide ageing and maximise repeatability and reproducibility of the experiment.

For negative controls, the primary antibody was omitted, as positive controls the internal normal tissues with known EpCam positivity were used. For each sample, staining intensity (0, faint to moderate, intense) and percentage of positive tumour cells was estimated. A case was considered Drug_discovery strongly positive if the antibody detected >70% positive cells, otherwise weakly positive, or negative if no cells were stained.