Nature Essay

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Supplementary Methods Human Endometrial Carcinoma Samples Flash frozen endometrial tissue from type I endometrioid tumours of patients of ages 30–55 was obtained under sterile conditions after hysterectomy at Peking University Oncology Hospital. The samples were graded and staged according to the 1988 International Federation of Gynaecology and Obstetrics (FIGO) systems. None of the patients had received preoperative chemotherapy or radiation. In addition, samples of normal endometrium that were age-matched to the cancers were obtained from patients undergoing hysterectomy for benign gynaecological diseases. All studies were approved by the Ethics Committee of the Peking University Health Science Center. Immunohistochemistry and Image Analysis Paraffin sections of 5 μm from the uteri were made. A standard immunohistochemical technique was used to detect the intensity and distribution of PCNA and Ki-67 immunostaining. A mouse anti-PCNA antibody (18-0110, Zymed Laboratories, Inc., San Francisco, CA) at a 1:150 dilution and a monoclonal mouse anti-human Ki-67 antibody (08-0156, Zymed Laboratories) were used. Quantification of PCNA immunostaining was performed on the digitized images of systematic randomly selected fields of endometrial stroma, from which non-stromal elements (e.g., luminal and glandular epithelium) were interactively removed and analyzed separately. A Leica microscope connected to a computer using Colorvision software (Leica Imaging System Ltd. Cambridge, England) was used for the image analysis. All luminal and glandular epithelia as well as 10 fields of stromal cells were measured separately in each tissue section. By using colour discrimination software, the total area of positively stained cells (brown reaction product) was measured and expressed as a ratio of the total area of cell nuclei (brown reaction product + blue haematoxylin).

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