Yang et al. BMC Cancer 2010, 10:133 http://www.biomedcentral.com/1471-2407/10/133 implicated in lymphomagenesis [9,10] and oncogenesis in nonsmall-cell lung cancer of human [11]. This sug- gested that the Bmi-1 gene plays an important role in cell proliferation and tumor progression. It has been confirmed that Bmi-1 gene is widely expressed in diverse human tumors, including non-small cell lung cancer, hepatocellular carcinoma, B-cell non-Hodgkin’s lymphoma, breast cancer, ovarian cancer, colorectal can- cer, skin cancer and neuroblastoma [10-20], and has been shown to be a useful prognostic marker in myelo- dysplastic syndrome and many cancers, including naso- pharyngeal carcinoma, bladder cancer and gastric cancer [17-20]. To date, however, the status of Bmi-1 expression and its clinical/prognostic relevance in ovarian cancer have not been fully elucidated. In this study, the protein expression and amplification status of Bmi-1 in a series of human epithelial ovarian tissue, normal and patholo- gical, non-neoplastic and neoplastic, were examined. The clinico-pathological and prognostic significance of expression of Bmi-1 in our ovarian carcinoma cohorts was also assessed. Methods Patients and tissue specimens In this study, a total of 249 epithelial ovarian tumors (benign, borderline and carcinomatous) were obtained from archives of paraffin-embedded tissues between 1996 and 2008 at the Department of Pathology, Cancer Center and the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China. The cancer cases selected were based on availability of resection tissue and follow- up data. Patients whose cause of death remained unknown were excluded from our study. The ovarian tumor cases encompassed 179 histologically confirmed invasive carcinomas, 40 borderline tumors and 30 cysta- denomas. Data of survival time and clinico-pathological parameters were collected. Ages of the 179 patients with ovarian carcinoma ranged from 18 to 86 years (mean age, 50.7 years) and their clinico-pathological character- istics are summarized in Table 1. None of the cancer patients in this study had received preoperative radiation or chemotherapy. In addition, 30 specimens of normal ovaries from exairesis for non-ovary diseases in the Department of Gynaecology and Obstetrics of the First Affiliated Hospital, Sun Yat-Sen University from 2005 to 2008 were used as control. For the use of these clinical materials for research purposes, prior patient’s consent and approval from the Institute Research Medical Ethics Committee of Sun Yat-Sen University was obtained. Construction of tissue microarrays (TMA) The TMA was constructed according to a method described previously [21]. Briefly, the individual donor Page 2 of 9 tissue block and the corresponding histological H&E stained slides were overlaid for tissue TMA sampling. The tissues (179 ovarian carcinoma, 40 borderline tumor, and 30 cystadenoma tissues) were sampled using a tissue arraying instrument (Beecher Instruments, Silver Spring, MD); a 0.6-mm-diameter cylinder of tissue was removed. Subsequently, the tissue cylinder was re- embedded into a predetermined position in a recipient paraffin block. In our constructed ovarian tumor tissue- TMA, 3 cores of sample were selected from each tumor tissue. Multiple sections (5 μm thick) were cut from the TMA block and mounted on microscope slides. Immunohistochemistry (IHC) IHC studies were performed using a standard streptavi- din-biotin-peroxidase complex method [22]. For antigen retrieval, tissue slides were microwave-treated and boiled in a 10 mM citrate buffer (pH 6.0) for 10 min. The slides were incubated with rabbit monoclonal anti- body against human Bmi-1 (Shangying Biotechnology Inc. Wuhan, China, 1:2000 dilution), mouse monoclonal antibodies against human p16Ink4a (1:200 dilution) or p14ARF (1:300 dilution, Labvision Co., Neomarkers, USA) overnight at 4°C in a moist chamber. A negative control was obtained by replacing the primary antibody with normal rabbit or mouse IgG. Known immunostain- ing positive slides were used as positive controls. Expression levels of Bmi-1 protein were visualized by observing the stained tissues under a light microscope. Positive expression of Bmi-1 was defined as the presence of brown or yellowish brown granules in the nuclei, though occasionally yellowish brown granules could also be seen in the cytoplasm. For evaluation of the Bmi-1 IHC staining in different ovarian tissues, a semi-quanti- tative scoring criterion for IHC of Bmi-1 described pre- viously [16] was used, in which both staining intensity and positive areas were recorded. A staining index (values 1 to 16), obtained as the intensity of Bmi-1 posi- tive staining (negative = 1, weak = 2, moderate = 3, or strong = 4 scores) and the proportion of immunoposi- tive cells of interest (≤ 10% = 1, >10% to ≤ 50% = 2, >50% to ≤ 75% = 3, >75% = 4 scores) were calculated. The two different scores of corresponding sample were then multiplied. Points equal or less than 4 was marked as (-); 4 points to 8 points was marked as (+); 8 points to 12 points, (++); and 12 points to 16 points, (+++). For statistical analysis, (-) and (+) were counted as low expression of Bmi-1 (Fig. 1A, B and 1C), while (++) and (+++) were counted as intensive expression of Bmi-1 (Fig. 1D and 1E). The evaluation of p16Ink4a and p14ARF IHC staining was scored on a semi-quantitative scale [14], as follows: negative expression (<10% of the cells were positive), down-regulated expression (small cell clusters, but 10-50% of the cells were positive) and