Journal of Experimental & Clinical Cancer Research 2008, 27:51 http://www.jeccr.com/content/27/1/51 Assessment of immunohistochemical staining Two pathologists, blinded to the clinicopathological data and patients' outcomes, independently evaluated immu- noreactivity of the tissue microarray slides. Discrepancies were resolved by simultaneous re-examination of the slides by both investigators. Immunostaining was evalu- ated by recording the percentage of cells staining and scor- ing the area of maximum staining on a 4-point scale, with 0 representing no staining and 3 representing the highest staining. The results from 3 cores in the same patient were averaged to obtain a mean value for subsequent statistical analysis. According to the median percentage of positive cells for each antibody, the protein expression results were classified into 3 groups: Negative group; Low group (less than the median value) and High group (greater than the median value). The median values were: 40% for p27, 10% for Skp2, and 20% for Cks1. As described in previous studies [13], the expressions of Skp2 and Cks1 was further stratified as either positive or negative (Low+High group versus Negative group), while the p27Kip1 expression was further classified as high expression (High group) and low expression (Negative+Low group). Statistical analysis Statistical data analyses were performed using SPSS 11.0 statistical software package (SPSS Inc., Chicago, IL). First, the associations among Skp2, Cks1, p27Kip1 and clinico- pathological factors were explored using χ2 test or Fisher's exact test as appropriate. Correlations between variables were tested according to the Spearman correlation test. Recurrence-free survival (RFS) and cancer-specific survival (CSS) were analyzed by Kaplan-Meier curves. The prog- nostic role of different parameters in the disease recur- rence and patient death was analyzed with the log-rank test. For multivariate testing, a stepwise forward Cox's pro- portional hazards regression model was performed to define the risk factors for tumor recurrence and patient death. P values less than 0.05 were considered significant. Two-sided tests were used throughout all the analyses. Results Immunohistochemical expression and its correlation with clinicopathological variables Representative examples of reactivity for p27Kip1, Skp2, and Cks1 are shown in Figure 1. In nonneoplastic renal tissue, the distal tubules, glomeru- lar epithelial cells, and pelvic urothelium showed strong nuclear p27Kip1 immunoreactivity but lacked immunore- activity for Skp2 and Cks1. The protein expression and clinicopathological data of the patients are summarized in Table 1. As shown in Table 1, Skp2, Cks1, and p27Kip1 expressions were not associated with patients' age or gender, as well as the ECOG-PS. In addition, none of the protein expression was associated with the grade. P27Kip1 expression was noted in 357 cases (74%), and high expression of p27Kip1 was identified in 168 cases (37%). The expression of p27Kip1 was significantly higher in clear-cell RCC compared with other histological types (P = 0.008). Furthermore, the expression significantly decreased with increasing tumor stage (P = 0.002) and tumor size (P < 0.001). In addition, the p27Kip1 was also significantly downregulated in cases with tumor necrosis (P = 0.001). And the expression was not significantly dif- ferent between primary and metastatic tumors (P = 0.062). Skp2 expression was noted in 68 of 384 (18%) clear cell and 3 of 81 (4%) papillary tumors, but none of chromo- phobe tumors. Skp2 expression was significantly corre- lated with tumor stage (P < 0.001) and tumor size (P < 0.001), as well as tumor necrosis (P = 0.001). But Skp2 expression was not significantly different between pri- mary and metastatic tumors (P = 0.136). Cks1 expression was similar to Skp2, noted only in 82 of 384 (21%) clear cell and 8 of 81(10%) papillary tumors. Among various clinicopathological variables, the expres- sion of Cks1 was only correlated with tumor size (P = 0.018). Associations among P27Kip1, Skp2 and Cks1 protein expression A strong correlation was found between Skp2 and Cks1 expression (P < 0.001), both of which were inversely related to p27Kip1 levels (P = 0.006 and P < 0.001). A fur- ther test was performed between patients with different metastatic status and histologic types. In primary RCCs, relation between Skp2 and Cks1 expression and their inverse relations to p27Kip1 levels are statistically signifi- cant (P < 0.001, P = 0.001 and P = 0.001, respectively). But in metastatic RCCs, no correlation was found: 5 Skp2-pos- itive RCCs showed Cks1 positive expression, while 5 Skp2-positive and 10 Cks1-positive RCCs showed low p27 expression, with a P value of 0.058, 0.179 and 0.893, respectively. In clear-cell RCCs, the correlation among the protein expression was the same as that in the whole patients. But in papillary tumors, only the inverse associations between p27Kip1 and Cks1 was statistically significant (P = 0.027). Survival analysis At the end of follow-up, 131 patients died due to cancer progression, 36 patients died of unrelated causes, and 128 patients developed disease recurrence. Page 3 of 8 (page number not for citation purposes)