Establishment of prediction system for the prognosis of patients with bladder cancer

(目的)在日本，膀胱癌的数量正在逐渐增加。然而，膀胱癌进展的分子机制尚未完全清楚。我们试图确定导致膀胱癌进展的基因(方法和患者)。在这项研究中，我们招募了84名2001年至2005年在我们附属医院接受经尿道膀胱肿瘤电切术(TURBT)和根治性膀胱癌切除术的患者。在获得所有患者的知情同意后，我们利用DNA微阵列分析了14例膀胱癌患者肿瘤进展的相关基因。为了进行这一分析，我们使用了ACE基因寡核苷酸阵列(日立软件工程有限公司，横滨)安装了30144个基因。我们还对肿瘤组织标本中的一些候选基因进行了免疫组织化学染色。用甲基化特异性聚合酶链式反应(MSP)检测候选基因启动子区域的甲基化状态。结果基因芯片检测到21个基因为…与正常膀胱组织相比，膀胱癌组织中上调的基因较多，下调的基因有18个。然后，我们通过实时定量聚合酶链式反应确认这些基因的上调或下调状态。因此，我们挑选了两个基因，S期蛋白相关蛋白2(Skp2)和细胞周期蛋白依赖蛋白1亚基1(CKS1)，作为膀胱癌进展过程中上调的基因。免疫组织化学研究表明，Skp2和CKS1在每个肿瘤标本中的表达相似，而p27的表达与这些基因的表达呈负相关。Kaplan-Meier分析显示，Skp2或CKS1阳性表达的患者预后明显差于相应的患者。MSP分析显示浸润性膀胱癌中p16INK4a和p14ARF基因甲基化率显著高于浅表性膀胱癌。Kaplan-Meier分析表明，p14ARF甲基化患者的预后明显比相应患者差。(结论)本研究提示，检测Skp2和CKS1的表达水平以及p16INK4a和p14ARF的甲基化状态对预测膀胱癌的进展和选择治疗方案有一定的参考价值。

(Purpose) The number of bladder cancer is gradually increasing in Japan. However, the molecular mechanism of bladder cancer progression has not yet fully understood. We attempted to identify genes responsible for tumor progression bladder cancer(Methods and Patients) In this study we enrolled 84 patients who received transurethral resection of bladder tumor (TURBT) and radical cystectomy between 2001 and 2005 in our affiliate hospitals. After obtaining informed consent from all patients, we analyzed the responsible genes for tumor progression in 14 patients with bladder cancer by DNA microarray. For this analysis, we used Ace gene Oligo array (Hitachi Software Engineering Co. Ltd, Yokohama) mounted 30144 genes. We also performed immunohistochemical staining for some candidate genes in tumor tissue specimens. Furthermore, methylation status of promoter region in some candidate genes was examined by methylation specific PCR (MSP)(Results) DNA microarray assay revealed that 21 genes were … More up-regulated and 18 genes were down-regulated in bladder cancer specimens compared with normal bladder tissue. We then confirmed the up-regulation status or down-regulation status of these genes by real-time PCR. Consequently, we picked up 2 genes, S-phase kinase-associated protein 2 (SKP2) and cyclin-dependent kinase subunit 1 (CKS1), as up-regulated genes during the process of bladder cancer progression. The immunohistochemical study demonstrated that expression of SKP2 was similar to that of CKS1 in each tumor specimen, while the expression of p27 was negatively correlated with the expression of these genes. Kaplan-Meier analysis demonstrated that patients with positive expression of either SKP2 or CKS1 had significantly poorer prognosis than the counterparts. MSP analysis revealed that methylation rates of p16INK4a and p14ARF were significantly higher in invasive bladder cancer than those in superficial bladder cancer. Kaplan-Meier analysis demonstrated that patients with p14ARF methylation had significantly poorer prognosis than the counterpart.(Coclusions) Our study suggest that determination of expression levels of SKP2 and CKS1 and methylation status of p16INK4a and p14ARF is useful for prediction of tumor progression and treatment selection in patients with bladder cancer Less