So far, there are very few results on endometrial cancer cells and Gd or GdA and no clinical data on endometrial cancer. Therefore, the aim of this study was to assess the expression of Gd on mRNA and protein level. Further, we aimed to spe cify the proportion of the immunosuppressive glyko modification GdA in tissue sellckchem samples of a large cohort of endometrial cancer patients by using an extensively vali dated anti GdA antibody. Finally, we aimed to analyse the impact of Gd/GdA positivity on clinical and patho logical features including patient outcome. Methods Patients Formalin fixed paraffin embedded tissue of 292 endometrial cancer patients was available. Most patients presented with early stage disease at pri mary diagnosis. 72. 6% of patients showed a Type I carcinoma with endometrioid histology.

Among the remainder there were 7. 9% with serous, Inhibitors,Modulators,Libraries 4. 1% with mucinous, 1. 7% with clear cell histology and 0. 3% with squamous cell histology. 11. 6% were classified as mixed and 1. 7% as undifferentiated carcinomas. Patients were also evaluated for concomitant Inhibitors,Modulators,Libraries diseases and pre sented with hypertension in 39. 7%, obesity in 30. 5% and diabetes in 11. 3% of all patients. Assay methods Immunohistochemistry Immunohistochemical staining has been described previously by us. Glycosylation dependant staining differences were assessed using the polyclonal Gd and the monoclonal GdA antibody. Specificity of GdA binding was analyzed by Western blot analysis. This antibody is suitable for the detec tion of GdA in endometrial tumour tissues.

Our former investigation showed that A87 B/D2 seems to be less restricted to GdA carbohydrate structures than other monoclonal antibodies made in our laboratories, although none of the three monoclonal antibodies recog nize GdS or other pregnancy Inhibitors,Modulators,Libraries related Inhibitors,Modulators,Libraries glycoproteins such as hCG or transferrin isolated from amniotic fluid. Formalin fixed paraffin embedded tissue Inhibitors,Modulators,Libraries sections were dewaxed with xylol and endogenous peroxidase activity was quenched by dipping in 3% hydrogen peroxide in methanol for 20 min. Then sections were rehydrated in descending concentra tions of alcohol. For GdA staining epitope retrieval was performed in a pressure cooker using sodium citrate buffer. Following PBS washes samples were blocked as described in Table 2 and incubated with the primary antibodies. Then samples were fur ther processed as per manufacturers instructions.

Finally, immunoreactivity was visualized using diaminobenzidine, slides were counterstained using haematoxylin, dehydrated in ascending concentra tions of alcohol, xylol treated and covered. Positive and negative controls were always included in the analysis. Preparation of LCL161? riboprobes Preparation of riboprobes was performed as described previously. In short, a 227 bp fragment of the Gd cDNA was cloned into the EcoR1 restriction sites of pBluescript SK and labelled with digoxi genin by in vitro transcription using the DIG RNA labeling Kit.