Journal of Experimental & Clinical Cancer Research 2008, 27:51 http://www.jeccr.com/content/27/1/51 formance status, pathological stage, tumor size, nuclear grading, and microscopic tumor necrosis. Yet, the accu- racy of the traditional clinical and histologic markers is still unsatisfactory in certain clinical settings. There lies the possibility that biologic markers, which have associ- ated with tumor progression, could serve as accurate prog- nostic markers or targets for specific intervention. As the alteration of cell cycle is a hallmark of cancer, pro- teins that are intimately involved in cell cycle regulation are of particular interest. The cell cycle progression is largely dependent on cyclins and cyclin-dependent kinases (Cdks) [3]. Cdks are regulated by Cdk inhibitors, including the INK4 family and the Cip/Kip family. The p27, a member of the latter (p27/Kip1), negatively regu- lates cell cycle by inactivating cyclin-CDK complex and preventing the transition from G1 to S phase. The degra- dation of p27 stimulates the activity of Cdk2/cyclin E and Cdk2/cyclin A to promote cell proliferation. Recent evi- dence also suggests that p27Kip1 is a putative tumor sup- pressor, thus the loss of p27Kip1 may lead to the uncontrolled proliferation of malignant cells [4]. Recently, reduced expression of p27Kip1 protein has been proved to be highly associated with tumor progression and poor prognosis in various malignant diseases [5]. However, downregulation of p27Kip1 mRNA is rarely observed in human cancers [6]. Instead, the decrease in p27Kip1 levels results mainly from ubiquitin-mediated proteolysis, regulated by the F-box protein SKP2(S-phase kinase-associated protein 2), and its cofactor, Cks1 [7]. SKP2 is an important component of the Skp1-Cullin-F- box protein (SCF) complex, which functions as the main rate-limiting regulator for the degradation of p27Kip1. Hence, overexpression of Skp2 may lead to cell-cycle pro- gression. Recent studies have also found that Skp2 may modulate invasion of cancer cells independent of p27 degradation [8]. Cks1 is a member of the highly conserved Cks/Suc1 proteins family, which confers an allosteric change in Skp2 to increase its affinity to phosphorylated p27Kip1 substrate [9,10]. Therefore, p27Kip1 degradation is dependent upon the accumulation of Skp2 and Cks1 as well as the rise in cyclin E. Recently, the expression levels of p27Kip1, Skp2 and Cks1 were shown to be highly asso- ciated with prognosis in a variety of cancers [10-13]. To date, very few studies have addressed the prognostic role of P27Kip1and Skp2 in renal cell carcinoma. And no study has elucidated the roles of Cks1 in RCCs. By using tissue microarray, we therefore aimed at analyzing the immunohistochemical expression patterns of p27Kip1, Skp2, and Cks1 proteins, and their associations with clin- ical and pathologic factors, as well as the prognostic implications. Methods Patients and specimens Our study cohort consisted of 482 patients who under- went radical or partial nephrectomy for RCC at the Shan- dong Provincial Hospital between 1993 and 2005. As approved by the ethical committees of Shandong Provin- cial Hospital, Formalin-fixed and paraffin-embedded specimens of these cases were chosen for analysis. Clinical data were recorded, including age, sex, and Eastern Coop- erative Oncology Group (ECOG) performance status (PS) score. Pathological variables were re-evaluated by 2 pathologists separately, including pT stage, Fuhrman grade and histological subtype. Patients were staged according to the 1997 staging system of the Union Inter- national Contre le Cancer (UICC) and American Joint Committee on Cancer (AJCC) [14]. The histological sub- types were stratified using Heidelberg classification [15] and the nuclear grade of tumors was determined by e Fuhrman grading scheme [16]. Performance status was determined using the Eastern Cooperative Oncology Group Performance Score (ECOG-PS) scale. The patients consisted of 329 men and 153 women, aging 19 to 94 years old, with a mean age of 62.1. There were 384 con- ventional, 81 papillary, and 17 chromophobe cases of RCCs respectively. 45 patients had distant metastases (to the lung in 12 patient, bone in 4 patients, the lymph nodes in 14 patients, gastrointestinal tract in 7 patients, soft tissue in 2 patients and adrenal gland in 6 patients) at the time of surgery. 324 (67.2%) tumors were discovered incidentally, 126 (26.1%) were locally symptomatic and 32 patients (6.6%) had systemic disease symptoms. The median period of follow-up for all patients was 62 months, ranging from 13 to 185 months. Construction of tissue microarray blocks and immunohistochemistry For immunohistochemical evaluation, tissue microarrays were constructed using a manual tissue arrayer with acces- sory (Beecher, Silver Spring, MD). 3 cylindrical core biop- sies (0.6 mm in diameter) were taken from different sites of each tumor and precisely arrayed in a recipient paraffin tissue microarray block. 10 specimens of non-neoplastic renal tissue resected from adjacent regions of renal cell carcinoma were also analysed for comparison. The procedures of immunohistochemical studies were performed as described previously [13]. In brief, 4-mm tissue microarray sections were incubated with the mouse monoclonal antibodies targeting Skp2 (1:100, Zymed), Cks1 (1:250, Zymed), p27Kip1 (1:50, Santa Cruz), using an automated immunostainer(Leica AutoStainer XL,). Bind- ing of the primary antibody was assessed using the DAKO EnVision kit (DAKO Corp.), and the hematoxylin served as counterstain. Incubation without the primary antibody was used as a negative control. Page 2 of 8 (page number not for citation purposes)