Journal of Experimental & Clinical Cancer Research 2008, 27:51 http://www.jeccr.com/content/27/1/51 Table 2: Univariate and Multivariate Survival Analysis P value on CSS P value on RFS Variables Univariatea Multivariateb Univariatea Multivariateb Age Gender TNM stage Tumor size Grade Histology ECOG-PS Necrosis p27Kip1 Skp2 Cks1 0.556 0.598 <0.001 <0.001 0.388 0.859 0.046 <0.001 <0.001 <0.001 0.002 0.236 0.326 <0.001 <0.001 0.078 0.396 0.874 0.117 0.003 0.360 0.110 0.522 0.885 <0.001 <0.001 0.085 0.704 0.259 <0.001 <0.001 0.001 0.029 0.413 0.590 <0.001 <0.001 0.219 0.730 0.427 0.242 0.002 0.966 0.482 Note: OS: overall survival; CSS: cancer-specific survival; RFS: recurrence-free survival. a Statistical analyses were performed by the log-rank test. b Statistical analyses were performed by Cox proportional-hazards regression model. p27Kip1 expression patterns among various histologic sub- types, such results were not confirmed in other studies [13]. In our study, the expression of p27Kip1 was signifi- cantly higher in clear-cell RCC compared with other histo- logic types and inversely related to TNM stage and tumor size. And no association between p27Kip1 expression and metastatic status confirms the result in previous study for RCC [13] and other carcinomas [23]. More intriguingly, the controversy regarding association between grade and p27Kip1 expression arised not only in RCCs, but also in many other cancers [24,25]. This dis- crepancy might be ascribed to the following reasons. First, the assessment of the grade may be highly observer dependent. Second, insufficient formalin fixation in some specimens may result in artificial reduced labeling of p27Kip1 in TMA. More likely, the root cause might stem from the complex regulatory mechanisms of p27Kip1 abundance. For instance, a new ubiquitin ligase for p27Kip1 has recently been identified, which is a member for Skp2-independent down-regulation of p27Kip1 at the G0–G1 transition [26]. The regulation of p27Kip1 is mainly determined by its rate of degradation rather than by transcription or translation. Recent studies indicate that Skp2 and Cks1, the cell-cycle regulatory proteins, play essential roles in the degradation of p27Kip1. Skp2 is an ubiquitin ligase responsible for tar- geting P27Kip1 to the proteasome and Cks1 is an essential cofactor for efficient Skp2 dependent p27Kip1 ubiquitina- tion. Several studies have provided evidence that both the Skp2 and Cks1 may have important roles in the develop- ment of tumor aggressiveness and its expression may be used as an independent prognostic factor for survival in various carcinomas[12,13,17,27]. In this study, Skp2 expression was found to be significantly correlated with advanced T stage, larger tumor size and necrosis, while Cks1 only correlated with tumor size. The results imply their importance in disease progression and confirm the previous studies. However, the different expression pat- tern among histologic types, such as no expression of Skp2 and Cks1 in chromophobe RCC, has never been pre- viously reported. Thus, the expression of Skp2 and Cks1 combined with p27Kip1 could help to separate the chromophobe from the clear cell subtype. As for the association among p27Kip1, Skp2 and Cks1, it is still in dispute [10-13]. In this study, there was a signifi- cant positive correlation between SKP2 and CKS1 expres- sion, as well as an inverse relationship between p27Kip1 and the 2 proteins. To examine whether other factors affected the association, we carried out further subanalysis dividing patients into subgroups based upon histologic subtypes and metastatic status. The correlation among these proteins was only found to be significant in primary and clear-cell RCCs. The observation may partially explain the current dispute, as the different samples used by differ- ent researchers. It also suggests that p27Kip1 downregula- tion may involve several pathways and occur through corresponding pathway in different status. Consistent with previous studies [13,20-22], we found that TNM stage and tumor size were strong predictive prognostic markers in patients with RCC. With regard to prognostic significance of protein expression, our study also demonstrated that low p27Kip1 expression signifi- cantly correlated with a poor outcome. Although the Skp2 and Cks1 expression correlated within inferior CSS and DFS in our univariate analysis, it did not remain prognos- tically independent in multivariate comparison. Conclusion In summary, our study suggests that immunohistochemi- cal expression levels of p27Kip1, Skp2 and Cks1 may serve as markers with prognostic implication. The inverse asso- ciation between p27Kip1 and Skp2 or Cks1 indicates the important roles of Skp2 and Cks1 in targeting the destruc- tion of p27Kip1, and these regulatory proteins may well be considered as novel targets of therapeutic intervention in RCC in the future. Competing interests The authors declare that they have no competing interests. Authors' contributions ZL and QF performed experiments and wrote the manu- script. JL has made substantial contributions to concep- tion and design, FW and KD has made substantial Page 7 of 8 (page number not for citation purposes)