应用定量PCR方法动态检测膀胱癌患者尿脱落细胞中survivin mRNA的表达及其临床意义

背景与目的：膀胱癌是泌尿系统最常见的恶性肿瘤,目前临床上缺乏理想的早期诊断方法。本研究通过检测膀胱移行细胞癌（bladder transitional cell carcinoma,BTCC）患者手术前后尿脱落细胞中survivinm RNA的表达,探讨其在BTCC患者早期诊断和术后监测肿瘤复发中的意义。方法：采用实时荧光定量PCR（real-time PCR）法检测10例健康志愿者、15例膀胱炎患者和30例初发BTCC患者术前及术后1周、1个月、6个月定期随访至15个月尿脱落细胞中survivin mRNA的表达情况。结果：30例BTCC患者术前尿脱落细胞中survivin mRNA的相对拷贝数为96．01±42．33。明显高于正常对照组及膀胱炎组（P〈0．05）。术后1周（25．30±1．51）较术前显著下降（P〈0．05）;术后1个月（13．20±1．49）、术后6个月（13．90±1．36）与正常对照组比较差异均无统计学意义（P〉0．05）;随访至15个月,3例复发患者复发时的相对拷贝数为97．83±27．47,与术后6个月相比差异有统计学意义（P〈0．05）。结论：尿脱落细胞中survivin mRNA的表达作为诊断膀胱移行细胞癌的指标敏感性高,术后动态随访survivin mRNA表达变化可监测复发。     

联合检测UBC、HA和CK20诊断膀胱癌的临床价值

背景与目的:膀胱癌是最常见的泌尿系统肿瘤,尿脱落细胞学检查是无创性诊断的金标准,但敏感性较低.本研究探讨联合运用尿膀胱癌抗原(urinary bladder cancer,UBC)、透明质酸(hyaluronic acid,HA)和细胞角蛋白20(cytokeratin 20,CK20)诊断膀胱癌的临床价值.方法:对64例膀胱癌患者、20例泌尿系良性疾病患者,在膀胱镜检查之前留尿,分别采用酶链免疫吸附实验、放射免疫分析和逆转录聚合酶链反应检测UBC、HA和CK20在尿液中的表达,同时行脱落细胞学检查,分析比较4种方法诊断膀胱癌的临床价值.结果:UBC、HA和CK20诊断膀胱癌的敏感性分别为85.9%(55/64)、89.1%(57/64)、78.1%(50/64),与脱落细胞学(40.6%)检查比较,差异有统计学意义(P<0.01);4种方法诊断膀胱癌的特异性分别为85,0%(17/20)、80.0%(16/20)、80%(16/20)和95%(19/20).各分级和分期肿瘤UBC、HA和CK20的敏感性分别高于尿脱落细胞学检查,UBC值各分级和分期比较差异无统计学意义(P>0.05).HA检测G2、G3组较G1组明显增高(P<0.01),但G2、G3组间比较差异无统计学意义(P>0.05);各分期之间比较差异无统计学意义(P>0.05).CK20检测随肿瘤的分级与分期的增高,敏感性增高(P<0.01).联合运用UBC、HA和CK20敏感性可达96.9%,特异性达100%.结论:联合UBC、HA和CK20能提高诊断膀胱癌的敏感性和特异性,初步诊断能够代替膀胱镜检查.     

Evaluation of two new urinary tumor markers: bladder tumor fibronectin and cytokeratin 18 for the diagnosis of bladder cancer.

Our objectives were to evaluate the diagnostic value of two new urinary tumor markers, cytokeratin 18 (CK18) and bladder tumor fibronectin (BTF), for the detection and monitoring of bladder cancer. The study comprised 931 urine samples belonging to 402 subjects: 112 individuals under suspicion for a primary bladder tumor (group 1); 104 bladder cancer patients under scheduled follow-up (group 2); 109 bladder cancer patients receiving intravesical instillations (group 3); 45 patients with other urological diseases (group 4); and 32 healthy subjects (group 5). Voided urine samples were collected before cystoscopies, between them and before intravesical instillations. CK18 and BTF tests were measured by chemiluminescent immunoassays. Optimal receiver operating characteristic cutoffs of 7.4 microg/L for CK18 and 52.8 microg/liter for BTF rendered overall sensitivities of 66.2% for CK18 and 80.0% for BTF at specificities of 88.4 and 74.7%, respectively. Urinary cytology provided a sensitivity of 29.2% at a specificity of 99.1%. Sensitivities were 80.8, 74.2, and 82.3% for BTF and 71.1, 77.4, and 64.7% for CK18 for groups 1 to 3, respectively. False positive rates were higher for BTF in all groups of patients. Elevated urinary tumor markers during the monitoring of patients with bladder cancer could detect recurrence sooner than scheduled cystoscopies. Persistence of negative markers was greatly indicative of free of disease status in follow-up. CK18 and BTF in urine may eventually prove to be of benefit for specific patients with bladder carcinoma given its higher sensitivity compared with cytology. In selected patients, namely those with persistent negative urinary CK18 and BTF, the number of cystoscopies could be reduced.     

Diagnostic value of fibronectin and mutant p53 in the urine of patients with bladder cancer: impact on clinicopathological features and disease recurrence

Development of new methods for bladder cancer detection is required because cystoscopy is invasive, and voided urine cytology (VUC) has low sensitivity. The aim of this study was to evaluate the diagnostic performance of urinary fibronectin and mutant p53 in comparison with VUC in the detection of bladder cancer. This study included 100 patients diagnosed with bladder cancer, 93 patients with benign urological disorders and 47 healthy volunteers. The urine supernatant was used for determination of fibronectin by ELISA, while urine sediment was used for cytology and detection of mutant p53 by PCR-SSCP followed by DNA sequencing. The sensitivity and specificity were 59% and 91.4% for VUC, 82% and 84.3% for fibronectin, and 37% and 100% for mutant p53; combination of the three parameters increased sensitivity to 95% but specificity was only 78.6%. A significant association was observed between disease recurrence and mutant p53, stage and lymph node involvement. Our results indicate that fibronectin had the highest sensitivity compared to VUC and mutant p53 in bladder cancer detection; however, mutant p53 had superior specificity compared to VUC and fibronectin. Mutant p53 is associated with disease recurrence and hence it has a significant prognostic role in bladder cancer.     

Urinary cell-free microRNA biomarker could discriminate bladder cancer from benign hematuria

The most common symptom of bladder cancer (BC) is hematuria. However, not all patients with hematuria are diagnosed with BC. Here, we explored a novel method to discriminate BC from hematuria under nonmalignant conditions by measuring differences in urinary cell-free microRNA (miRNA) expression between patients with BC and those with hematuria. A multicenter study was performed using 543 urine samples obtained from the National Biobank of Korea, including 326 BC, 174 hematuria and 43 pyuria without cancer. The urinary miR-6124 to miR-4511 ratio was considerably higher in BC than in hematuria or pyuria, and enabled the discrimination of BC from patients with hematuria at a sensitivity of >90% (p < 0.001). Conclusively, the proposed noninvasive diagnostic tool based on the expression ratio of urinary cell-free miR-6124 to miR-4511 can reduce unnecessary cystoscopies in patients with hematuria undergoing evaluation for BC, with a minimal loss in sensitivity for detecting cancer.     

人膀胱癌细胞角蛋白20 mRNA表达的研究

目的　评估细胞角蛋白 2 0mRNA(CK2 0mRNA)是否可以作为膀胱癌临床诊断的一种有用标志物。方法　对 84例肉眼血尿患者的尿液进行尿脱落细胞学及CK2 0mRNA标记物RT/PCR检测。分析参数包括肿瘤数目、大小及WHO分级 ,术前或活检前尿脱落细胞学和CK2 0mRNA标志物。结果　病理活检证实 2 2例移行细胞癌中 ,CK2 0mRNA 18例为阳性 ,4例阴性 ;6 2例非膀胱癌患者中 ,CK2 0mRNA标志物 2例假阳性。与尿脱落细胞学比较 ,CK2 0mRNA标志物对膀胱移行细胞癌诊断的特异性和阳性预报值更高 ,分别为 96 .8%比 77.4 % (U =3.2 1,P <0 .0 1) ,90 %比 5 1.7% (U =2 .81,P <0 .0 1) ;但两者在敏感性和阴性预报值间无显著性差异 (U值分别为 :1.0 4和 1.2 1,P >0 .0 5 )。CK2 0mRNA表达与肿瘤分级间无明显相关性。结论　通过RT/PCR方法检测CK2 0mRNA是诊断膀胱移行细胞癌的一种良好生物标志物 ,其特异性明显优于尿脱落细胞学。     

Increased levels of urinary basic fibroblast growth factor in bladder cancer patients

Urinary basic fibroblast growth factor (bFGF) was determined by a competitive sandwich ELISA test in 26 patients with transitional bladder cancer and 26 normal volunteers or subjects with benign urological diseases. The median bFGF value in the patient group was 3.41 ng bFGF/g creatinine (range: 0-153.6), significantly higher than the median levels in the control group (1.39 ng/g creatinine; range: 0-4.5). On the basis of the optimal cut-off point of 2.64 ng bFGF/g creatinine, the sensitivity of the test for detecting bladder cancer was 61.5% and the specificity 76.9%. We also studied 17 individuals successfully treated for a previous bladder cancer and with no evidence of disease at the moment of urine collection (NED group). These subjects showed similar urinary bFGF levels as those observed in the control group (median 0.24 ng bFGF/g creatinine, range 0-5.1). Our data suggest that the dosage of urinary bFGF could be a non-invasive useful assay in the management of bladder cancer patients.     

尿透明质酸含量诊断膀胱癌的临床研究

目的　探测尿液透明质酸 (HA)含量检测在膀胱移行细胞癌诊断中的意义并研究放免方法代替ELISA方法检测尿液中HA含量的可能性。方法　同时使用ELISA和放免两种方法对 49例膀胱移行细胞癌病人、12例良性膀胱肿瘤病人、30例BPH和泌尿系结石病人和 2 0例正常人尿液中HA含量进行检测。结果　ELISA方法和放免方法测得各组尿液HA值结果相近 (P >0 .0 5 )。以放免方法所得结果进行分析 ,膀胱移行细胞癌病人组尿液HA含量是良性膀胱肿瘤病人组、泌尿系非肿瘤性疾病病人组和正常成人组的 2 -4倍 ,差异性极显著 (P <0 .0 1)。以对照组尿液HA水平上限137.5ng/mg( 113 .6 + 2 3.9=137.5ng/mg )作为阳性界值时 ,尿液HA含量检测诊断膀胱移行细胞癌的敏感度为 91.8% ,特异度为 91.9% ,与尿脱落细胞学相比 (敏感度为 48.9%、特异度为 95 .2 % ) ,敏感度差异有极显著意义 (P <0 .0 1) ,特异度差异无统计学意义 (P >0 .0 5 )。结论　尿液HA含量检测是一种简单、无创、价廉、结果客观、敏感度和特异度均较高的诊断膀胱移行细胞癌的方法 ;价格更为低廉的放免法可代替ELISA方法进行尿液HA含量的检测。     

Ways to improve the early detection of bladder cancer patients (based on Grodno Province data)]

The authors present an analysis of the data on 281 primarily recognized patients with cancer of the urinary bladder in Grodno Province 1968--1974. Among all these patients 49.5% were recognized 1 year and longer following the initial clinical manifestations of the disease. In every fifth case there was a far-advanced process. The causes of late recognition are as follows: underestimation of a tainsiet hematuria by patients, and in a number of cases the absence of oncological vigilance among general practitioners in the treatment of patients showing hematuria. To improve the early diagnosis of the bladder cancer, it is suggested to differentiate cancer high-risk groups. This contingent should be periodically examined by a specialist with prophylactic purposes. Moreover, an attention is paid to the necessity of a detailed urological examination of all persons with hematuria, or who showed it previously.     

抗凋亡基因bcl-2启动子CpG岛的高甲基化状态见于28.3%(13/46例)的膀胱癌患者尿沉淀细胞DNA

目的:检测上海地区膀胱癌患者尿沉淀细胞中3个肿瘤相关基因启动子CpG岛的甲基化谱式异常频率,继而评估其应用前景。方法:采用甲基化特异性PCR方法分析尿沉淀细胞DNA中DAPK1、bcl2和hTERT3个基因的启动子CpG岛甲基化状态,并通过RTPCR方法评估DAPK1基因在膀胱癌细胞系中的表达状态。结果:对膀胱癌细胞系中DAPK1基因的启动子CpG岛甲基化状态及其表达(mRNA水平上)所做的分析,确立了高甲基化状态与表达静息化之间的相关性。对46例临床确诊的膀胱癌患者和84例非膀胱癌对照(包括前46例术后的36例)的尿沉淀细胞中DNA甲基化的分析发现,仅bcl2基因的高甲基化见之于28.3%(13/46例)的膀胱癌患者,而84例的对照中均为去甲基化状态。结论:在美国膀胱癌患者尿沉淀细胞中频发DNA高甲基化的靶点在上海地区膀胱癌患者人群中频率很低,因此寻找在后者中频发DNA高甲基化的新靶点实属必要。     

High-throughput molecular analysis of urine sediment for the detection of bladder cancer by high-density single-nucleotide polymorphism array

The detection of urothelial malignancies remains challenging. The majority of patients diagnosed with bladder cancer require life-long surveillance for disease recurrence. Monitoring strategies rely predominantly on invasive endoscopic techniques, which are inconvenient and uncomfortable. Multiple in vitro diagnostic technologies have been developed to supplant the contemporary standard of care. The U.S. Food and Drug Administration has approved several assays, but [because of inferior performance characteristics (low sensitivity and specificity)] these tests have not made a significant impact on practice, to date. We sought to develop a test for bladder cancer with better performance characterization detection based on a novel molecular approach. Matched urine and peripheral blood lymphocyte samples were obtained before surgery from 31 patients with bladder cancer (10 pTa, 4 pT1, and 17 pT2>or). DNA from these samples was subjected to allelic imbalance analysis using HuSNP chips and was validated in parallel with microsatellite analysis for loss of heterozygosity and microsatellite instability. Peripheral blood lymphocyte and urine DNA obtained from 14 individuals without clinical evidence of genitourinary malignancy served as controls. Thirty-one of 31 (100%) urine DNA samples from patients with bladder tumors were found to have 24 or more single-nucleotide polymorphism (SNP) DNA alterations. In general, SNP alterations were more common in urine samples from pT2>or tumors than pTa or pT1 tumors. SNP alterations were not identified in nine normal control subjects and in four of five patients with hematuria. These data support the noninvasive HuSNP chip assay in urine DNA as a valuable tool for the detection of bladder cancer (on a high-throughput-automated platform).