Yang et al. BMC Cancer 2010, 10:133 http://www.biomedcentral.com/1471-2407/10/133 Page 3 of 9 Table 1 Association of Bmi-1 expression with patient’s clinico-pathological features in ovarian carcinomas Bmi-1 protein Low expression All cases Intensive expression Age at surgery (years) ≤ 50.7b > 50.7 Histological type Serous (grade 1) Serous (grade2/3) Mucinous Endometrioid Clear cell Undifferentiated Histological grade (Silveberg) G1 G2 G3 pT status pN status pM status FIGO stage aChi-square test bMean age pT1 pT2 pT3 pN0 pN1 pMX pM1 I II III IV 92 87 11 107 23 8 7 23 36 101 42 51 35 93 88 91 153 26 33 21 99 26 60 (65%) 52 (60%) 9 (82%) 68 (64%) 15 (65%) 7 (88%) 5 (71%) 8 (35%) 29 (81%) 63 (62%) 20 (48%) 39 (76%) 22 (63%) 51 (55%) 66 (75%) 46 (51%) 102 (67%) 10 (38%) 27 (82%) 17 (81%) 58 (59%) 10 38%) 32 (35%) 35 (40%) 2 (18%) 39 (36%) 8 (35%) 1 (12%) 2 (29%) 15 (65%) 7 (19%) 38 (38%) 22 (52%) 12 (24%) 13 (37%) 42 (45%) 22 (25%) 45 (49%) 51 (33%) 16 (62%) 6 (18%) 4 (19%) 41 (41%) 16 (62%) P valuea 0.452 0.038 0.011 0.037 0.001 0.006 0.002 diffuse expression (>50% of the cells were positive). Results were observed and assessed by two independent pathologic doctors, without knowing the identity of the samples. Fluorescence in situ hybridization (FISH) Two-color FISH was performed using a Spectrum Orange-labeled BAC clone (RP11-573G6) at 10p12 con- taining the Bmi-1 gene and a Spectrum Green-labeled reference centromeric probe on chromosome 10 (Vysis, Downers Grove, IL). The FISH reaction was performed as described previously [23] with slight modification. Briefly, deparaffinized ovarian carcinoma tissue sections were treated with proteinase K (400 μg/ml) at 37°C for 30 min, followed by denaturing in 70% formamide, 2× SSC at 75°C for 6 min. Fifty nanograms of each probe were mixed in a 20 μl hybridization mixture (containing 55% formamide, 2× SSC, and 2 ∝g human Cot1 DNA), denatured at 75°C for 6 min and then hybridized to the denatured tissue sections at 37°C for 24 hours. The slides were counterstained with 1 μg/ml DAPI in an anti-fade solution and were examined with a Zeiss Axio- phot microscope equipped with a triple-band pass filter. A minimum of 300 tumor cells was evaluated per speci- men. Amplification of Bmi-1 was defined as presence of either 6 (or more) Bmi-1 gene signals or at least 3 times as many gene signals than centromere signals of chro- mosome 10 in tumor cells (Fig. 1F). Statistical analyses Statistical analysis was carried out with the SPSS statisti- cal software package (SPSS Standard version 13.0, SPSS Inc.). The association of Bmi-1 protein expression with ovarian carcinoma patient’s clinico-pathological features was assessed by the Chi-square test. Multivariate survival analysis was performed on all parameters that were