1 3 p53 k-ras 4 5 6 8 9 11 12 13 14 15 16 17 18 19 20 21 22 Aurora-A 15 16 19 20 22 12 17 18 13 16 21 22 20 Patients and Methods Patients 1 23 24 The day of thoracotomy was considered the starting day for counting postoperative survival days. The median and mean follow-up periods were 1697 and 1983 days, respectively (range, 50–6381 days). This study has been approved by the Ethics Committee of Faculty of Medicine, Kyoto University. Tissue Preparation All tumor specimens were fixed immediately in 10 vol% formalin, and then embedded in paraffin. Serial 4-μm sections were prepared from each sample and used for routine hematoxylin and eosin (HE) staining, the TUNEL staining to detect apoptotic cells, and IHS to determine Aurora-A expression status, p53 status, intratumoral microvessel density (IMVD), and proliferative index (PI). Immunohistochemistry Aurora-A expression was evaluated using a standard streptavidin-biotinylated horseradish peroxidase detection system. Dewaxed sections were incubated overnight with a rabbit polyclonal antibody (KR051, Transgenic Co. Ltd., Kumamoto, Japan; it had been produced and its reliability had been confirmed by western blotting on a commercial basis) diluted at 1:100. Diaminobenzidine-tetrahydrochloride (0.03%) containing 0.1% hydrogen peroxide was used as a chromogen, and sections were counterstained with hematoxylin. For negative control slides, rabbit immunoglobulin G was used instead of the primary antibody. Colon cancer sections that are known to express Aurora-A were used as positive control slides. 1 B 1 C Fig. 1. A B C  23 25 23 25 23 Detection of Apoptosis 23 Statistical Analysis t U P Results Expression of Aurora-A in NSCLC n 1 Table 1. Characteristics of patients and Aurora expression  No. of patients (%) MAGE-D4 expression P Negative Positive Diffuse cytoplasmic Perimembrane All patients 189 (100) 31 (16.4%) 124 (65.6%) 34 (18.0%)  Age (mean ± SD) 62.4 ± 9.6 60.5 ± 10.2 62.3 ± 9.2 64.3 ± 9.1 0.256  Sex     .137     Male 133 (70.4) 26 (19.5%) 86 (64.7%) 21 (15.8%)      Female 56 (29.6) 5 (8.9%) 38 (67.9%) 13 (23.2%)  Performance status (PS)     .353     0 164 (86.8) 28 (17.1%) 109 (66.5%) 27 (16.5%)      1–2 25 (13.2) 3 (12.0%) 15 (60.0%) 7 (28.0%)  Histologic type     .302 (Sq vs Ad)     Squamous cell (Sq) 64 (33.9) 8 (12.5%) 41 (64.1%) 15 (23.4%)      Adeno (Ad) 108 (57.1) 19 (17.6%) 73 (67.6%) 16 (14.8%)      Large cell (La) 10 (5.3) 4 (40.0%) 4 (40.0%) 2 (20.0%)      Others 7 (3.7) 0 (0.0%) 5 (85.7%) 1 (14.3%)  *     .393     Well 68 (37.4) 9 (13.2%) 49 (72.1%) 10 (14.7%)      Moderately 67 (36.8) 13 (19.4%) 43 (64.2%) 11 (16.4%)      Poorly 47 (25.8) 9 (19.1%) 26 (55.3%) 12 (25.5%)  Pathologic (p-) stage     .007 (for all p-stage)     I 104 (55.0) 12 (11.5%) 80 (76.9%) 12 (11.5%)      II 22 (11.6) 4 (18.2%) 11 (50.0%) 7 (31.8%)      IIIA 63 (33.3) 15 (23.8%) 33 (52.4%) 15 (23.8%)  * 1 1 P 2 2 Table 2. Correlation between Aurora expression and other biomarkers  Aurora expression P Negative Positive Diffuse cytoplasmic Perimembrane * 191.2 ± 16.3 174.7 ± 8.9 167.6 ± 14.6 .584 Proliferative index (PI) (%) 49.2 ± 4.8 41.7 ± 2.5 63.5 ± 3.8 <.001 Apoptotic index (AI) (/1000 tumor cells) 13.5 ± 3.2 20.5 ± 2.0 15.9 ± 2.5 .154 p53-status: Aberrant expression (+) 12/31 (38.7%) 55/124 (44.4%) 15/34 (44.1%) .848 Values were shown as the mean ± standard error (SE). * Expression of Aurora-A and Postoperative Survival P 3 2 Table 3. Aurora expression and postoperative survival in non-small-cell lung cancer (NSCLC) patients  5-year survival rate (%) P AURORA expression Negative Positive Diffuse cytoplasmic Perimembrane All patients 67.8% 66.7% 47.6% .033 Subset analysis, histologic type     Squamous cell (Sq) 75.0% 74.2% 34.5% .007     Adeno (Ad) 64.2% 60.8% 62.5% .894 Pathologic (p-)stage     I 91.7% 72.9% 58.3% .098     II 75.0% 68.6% 57.1% .579     IIIA 50.4% 50.9% 28.9% .088 Fig. 2. Survival curves of patients who underwent complete resection for pathologic (p-) stage I–IIIA non-small-cell lung cancer. Comparison according to Aurora-A expression status.  P P 3 4 Table 4 Multivariate analysis of prognostic factors (Cox’s proportional hazard model) Factors Beta P Hazard ratio (95% confidence interval) Gender (male/female) −0.539 .096 0.583 (0.309–1.101) Age 0.006 .714 1.006 (0.977–1.035) Performance status (0/1/2) 0.183 .571 1.201 (0.637–2.264) Histologic type (Nonadenocarcinoma/adenocarcinoma) −0.050 .162 0.951 (0.887–1.020) Pathologic stage (I/II/IIIa) 0.551 .001 1.734 (1.311–2.295) Perimembrane Aurora expression (No/Yes) −0.362 .032 0.343 (0.169–0.699) Discussion 8 20 22 26 27 26 Aurora-A Aurora-A 22 22 13 15 18 20 15 16 20 17 18 13 16 20 2 3 15 16 17 20 3 8 26 2 28 30 28 29 30 22 p53 Aurora-A 31 36 31 35 32 33 34 Aurora-A