膀胱癌患者尿脱落细胞中Survivin的检测及其临床应用

目的通过对膀胱移行细胞癌（TCCB）患者尿脱落细胞中Survivin蛋白和其mRNA表达的检测，以探讨其在膀胱肿瘤早期诊断中的应用价值。方法分别采用免疫组织化学sP法和巢式逆转录-聚合酶链反应（Nested RT-PCR）方法检测48例TCCB、16例对照组（其中5例膀胱外泌尿系肿瘤患者、5例非肿瘤泌尿系疾病患者及6例健康者）尿脱落细胞Survivin的蛋白和mRNA表达，同时行尿脱落细胞学检查。结果48例TCCB患者中尿脱落细胞Survivin蛋白与mRNA阳性表达率分别为42例（87．5％），47例（97．9％）；对照组仅1例BPH患者mRNA阳性表达（3．1％）。TCCB组与对照组Survivin阳性率比较差异有统计学意义（P〈0.01）。免疫组织化学和RT—PCR法检测尿液中Survivin的敏感性和特异性分别为87．5％、97．9％和72．7％、93．8％。而尿脱落细胞学检查阳性率为28例（58．3％），其敏感性和特异性分别为58．3％和100．0％。结论尿脱落细胞Survivin检测诊断TCCB敏感性和特异性均较高，且无创，无痛苦，可作为早期诊断TCCB的敏感指标，其中RT-PCR检测方法敏感性更高。     

尿及癌组织中Survivin的表达对膀胱癌的早期诊断价值

目的 通过对膀胱移行上皮癌患者尿脱落细胞及癌组织中抑制凋亡相关基因Survivin的检测，探讨早期发现、常规筛选膀胱癌且无损伤的方法。方法留取53例膀胱移行上皮癌、20例泌尿系其他疾病患者及10例健康志愿者新鲜中段晨尿200mL，对53例膀胱移行上皮癌患者术后癌组织，用巢式逆转录聚合酶链反应（nestedRT-PCR）检测Survivin的表达，同时行尿脱落细胞学检查及膀胱镜取材病检。结果用巢式RT—PCR检测53例膀胱移行上皮癌患者尿及癌组织中Survivin均有表达，所有样本尿中Survivin的表达敏感性为100％，特异性为90％。尿脱落细胞学敏感性为37．7％，特异性为100％。膀胱镜病检敏感性、特异性均为100％。尿及癌组织中Survivin与尿脱落细胞学敏感性比较有显著差异（P〈0．05），与膀胱镜检相同。而特异性分别为90％、100％、100％，P〉0．05，无差异。结论用巢式RT-PCR方法检测膀胱移行上皮癌患者尿及癌组织中Survivin的表达，其敏感性、特异性相同。尿脱落细胞Survivin表达敏感性、特异性高。尿取材无损伤、方便，敏感性优于尿脱落细胞学检查，与膀胱镜检相当，特异性与尿脱落细胞学检查及膀胱镜病检无差别，且对膀胱癌前病变的归转有一定价值。检测尿中Survivin有望成为临床诊断、筛检膀胱癌的较可靠方法。     

尿Cox-2蛋白与CK20 mRNA诊断膀胱移行细胞癌的临床价值

目的：评价尿环氧化酶-2（Cox-2）蛋白、CK20 mRNA诊断膀胱移行细胞癌的临床价值。方法：选择78例膀胱移行细胞癌和30例非膀胱肿瘤患者，同时行尿Cox-2蛋白、CK20 mRNA和尿脱落细胞学检查,比较各种方法的敏感性、特异性和Youden指数。结果：尿Cox-2蛋白、CK20 mRNA和尿脱落细胞学的敏感性分别为89.7％．66.7％．26.9％；特异性分别为93.3％、86.7％和100％；Youden指数分别为83.1％、53.4％和26.9％。尿Cox-2蛋白和CK20 mRNA的敏感性和Youden指数均高于尿脱落细胞学（P＜0．05）,同时尿Cox-2蛋自的敏感性和Youden指数高于CK20 mRNA（P＜0．05）。结论：尿Cox-2蛋白和CK20 mRNA检测的高敏感性、高特异性为膀胱癌提供了一个简单无创的检测方法，非常适合于复发率很高的膀胱癌的诊断与随访。     

几种新瘤标对膀胱癌早期诊断价值的比较

目的：比较几种新瘤标对膀胱移行细胞癌早期诊断的价值。方法：对322例肉眼或镜检血尿、膀胱刺激症状或发现膀胱占位者，在作膀胱镜检查前行尿液中细胞角蛋白20(CK20)、核基质蛋白(NMP22)、ImmunoCyt、膀胱肿瘤抗原(BTA stat)检测和常规尿细胞学检查，比较它们的敏感性和特异性。结果：经膀胱镜和病理检查诊断膀胱移行细胞癌149例，其中浅表性肿瘤110例，浸润性39例；G181例，G246例．G322例。CK20、NMP22、ImmunoCyt、BTA stat、尿细胞学和膀胱镜检的敏感性分别为90．6％、85．2％、82．6％、65．8％、30．2％和94．6％，特异性分别为83．6％、84．0％、78．1％、64％、100％和100％。结论：新瘤标CK20、NMP22、ImmunoCyt具有较高的敏感性，与BTA stat和尿细胞学比较，差异有统计学意义，可用于膀胱肿瘤的早期诊断。     

survivin与nmp22对诊断尿路上皮肿瘤价值的比较

目的 :评价尿脱落细胞中survivin的表达和尿nmp2 2的表达对诊断尿路上皮肿瘤的价值。 方法 :对4 8例尿路上皮肿瘤患者 ,在行膀胱镜检查或手术前留新鲜尿液分别行尿脱落细胞survivin、尿nmp2 2和尿脱落细胞检查 ,并分别比较各方法的敏感性、特异性。结果 :尿路上皮肿瘤患者尿脱落细胞涂片中survivin表达 4 8例中4 6例阳性 ,尿NMP2 2有 38例阳性 ,而尿脱落细胞学仅 15例阳性 ,survivin的敏感性高于nmp2 2及脱落细胞学 (P <0 .0 5及P <0 .0 1)。三者的特异性分别为 10 0 %、90 %和 10 0 % ,差异无统计学意义。结论 :检测尿脱落细胞中survivin的表达方法简单无创敏感性、特异性高对诊断尿路上皮肿瘤的价值优于尿nmp2 2。     

核基质蛋白22在膀胱移行上皮癌诊断中的价值(附48例报告)

目的:评价患者尿中核基质蛋白22(NMP 22)在泌尿系上皮肿瘤诊断中的意义。方法:采用ELISA法测定48例膀胱移行上皮肿瘤患者尿中NMP 22的值,并与尿脱落细胞学检查进行比较。结果:48例膀胱移行上皮肿瘤患者尿NMP 22的中位值为19.53 IU/L。以10 IU/L为临界值,NMP 22诊断膀胱移行上皮肿瘤的敏感性为86.96％,特异性为50％;尿脱落细胞学检查的敏感性为17.39％,特异性为100％。尿NMP 22在肿瘤的分期、分级间的差别无显著性意义(P>0.05)。结论:尿NMP 22检测比尿脱落细胞学检查更敏感,可以作为血尿患者和既往膀胱肿瘤患者的首选筛选方法。     

荧光原位杂交技术在膀胱尿路上皮癌诊断中的临床应用

目的 探讨荧光原位杂交(FISH)技术运用于膀胱尿路上皮癌的诊断价值.方法 收集20例健康志愿者的新鲜晨尿,运用荧光标记的3号、7号、17号染色体着丝粒探针及9号染色体p16位点探针,对尿液标本中的脱落细胞染色体进行FISH技术检测,建立正常人群的阈值.收集158例怀疑为膀胱尿路上皮癌患者的新鲜晨尿,在行膀胱镜检查前,同期进行FISH技术与尿脱落细胞学检测,运用统计学方法,比较FISH技术与尿脱落细胞学检测的敏感性与特异性.结果 FISH与尿脱落细胞学的敏感性分别为84.8%和43.8%,FISH敏感性高于尿脱落细胞(P<0.05),FISH与尿脱落细胞学特异性分别为89.1%和87.0%,两者无统计学差异(P>0.05),在不同的肿瘤病理分级中,FISH的敏感性都高于尿脱落细胞,并且FISH敏感性随肿瘤分级逐级升高(P<0.05).结论 FISH技术具有较高的敏感性和特异性,可以作为国人膀胱尿路上皮癌筛查、诊断的新方法.     

膀胱肿瘤与尿液瘤标检测

膀胱肿瘤是泌尿系统最常见的恶性肿瘤，首次发现75％以上是浅表性膀胱肿瘤，其中10％～20％的病例会发展，故膀胱肿瘤早期诊断意义重大。传统的尿脱落细胞学检查阳性率较低，用特殊染色或免疫细胞染色对脱落细胞进行瘤标检测，可大大提高阳性率。同时对尿液中游离的瘤标进行检测，对膀胱肿瘤的诊断、预后判断、疗效评估也具有重要价值。现将这两种癌标检测方法综述如下。1尿脱落细胞瘤标检测1．1吖啶橙（AO）染色癌细胞DNA含量高于正常细胞，此变化早于形态学改变。AO作为荧光染料以插入方式与核酸特异性结合，可检测尿脱落细胞中DNA异…     

荧光原位杂交法在膀胱尿路上皮癌诊断中的应用

目的：探讨荧光原位杂交法（FISH）在膀胱尿路上皮癌诊断中的应用。方法：选取20例非尿路上皮癌和40例膀胱尿路上皮癌的人群尿液作常规尿脱落细胞学检查和FISH检测。结果：FISH技术的敏感性为82．5％,显著高于常规尿脱落细胞学的敏感性25．0％（P〈0．05）;FISH技术和常规脱落尿细胞学检查的特异性均为100％,两者在特异性方面差异无统计学意义（P〉0．05）。结论：荧光原位杂交法在膀胱尿路上皮癌诊断中的特异性与常规尿脱落细胞学检查一致,但其敏感性显著高于常规尿脱落细胞学检查,所以,FISH技术更有望成为膀胱尿路上皮癌无创性的诊断和检测手段。     

六位点微卫星组合用于膀胱癌诊断的研究

目的　探讨 6个微卫星位点组合在膀胱肿瘤诊断中的意义。　方法　选择 10个微卫星位点 (D9S16 2、D9S171、IFNA、D9S176、D9S195、D14S5 1、ACTBP2、D14S2 6 7、D17S6 95、D2 1S12 4 5 ) ,应用PCR SSLP法检测 32例膀胱肿瘤患者的肿瘤组织、膀胱冲洗液沉渣微卫星改变 ,15例非膀胱肿瘤患者为对照组。　结果　 32例膀胱肿瘤组织中 ,微卫星改变阳性 30例 ,敏感性为 93.8% (30 / 32 ) ,对照组膀胱冲洗液沉渣微卫星改变均为阴性。微卫星改变与膀胱肿瘤分期、分级无相关性。选取微卫星改变发生率最高的D9S171、IFNA、D14S5 1、D17S6 95、D2 1S12 4 5、ACTBP2 6位点组合微卫星改变阳性率为 90 .6 % (2 9/ 32例 ) ,与 10个微卫星位点组合相比差异无显著性意义。　结论　此 6个微卫星位点组合检测膀胱肿瘤可能具有较高的敏感性和特异性。     

Microsatellite alterations in human bladder cancer: detection of tumor cells in urine sediment and tumor tissue

OBJECTIVES: Bladder cancer is the result of clonal expansion of cancer cells in which multiple genetic alterations have occurred. Loss of heterozygosity (LOH) studies have demonstrated that alterations in microsatellite regions are common in bladder cancer. This observation offers the possibility of early tumor detection by examining the DNA of urinary sediment.METHODS: We investigated alterations of 17 microsatellite loci in urinary bladder carcinomas of different stages and grades. Per locus, 19-30 specimens were evaluated. DNA was isolated from tumor specimens, urinary sediment and peripheral blood lymphocytes. DNA fragments of 17 microsatellite loci were amplified by PCR and analyzed for genomic alterations.RESULTS: Microsatellite alterations were detected in tumor tissue and urine sediment from 27 out of 31 patients (87%). Urine sediment analysis alone proved positive in 24 out of 31 patients (77%). The type of lesions most frequently detected was LOH (74% of all alterations), followed by length alteration (24%) and additional alleles (2%). On average, the alteration frequency was 22% per locus. The loci at chromosomes 9 and 18 proved most informative. No alterations were found in grade I tumors. The study revealed a correlation between microsatellite alterations and the respective grades and stages of the tumors. Average alteration frequencies per locus were: 27.4% in grade III versus 19.3% in grade II tumors, 26.5% in invasive versus 12.3% in superficial tumors.CONCLUSIONS: Our results demonstrate that microsatellite alterations are common in bladder cancer and that analysis of genomic instabilities in urine samples should be further evaluated as a method for bladder cancer screening in a high-risk group. Especially, when a set of microsatellites is used that shows a high probability of detecting alterations and allows easy handling, this could be an alternative or a completion to currently available methods.     

Increase sensitivity in detecting superficial,low grade bladder cancer by combination analysis of hypermethylation of E-cadherin,p16, p14, RASSF1A genes in urine

ObjectivesTo identify a better set of DNA methylation markers to detect superficial, low grade cancer cell in urine sediment for improving cancer treatment, morbidity, and mortality.Materials and MethodsMethylation-specific PCR (MSP) assay was used to detect promoter hypermethylation in 4 genes (E-cadherin, p16, p14, and RASSF1A) to identify reliable biomarkers for bladder cancer diagnosis in primary tumor DNA and urine sediment DNA from 57 bladder cancer patients. Urine DNA was compared with 20 healthy controls.ResultsFifty-one (90%) tumor DNA and 47 urine DNA (83%) samples from bladder cancer patients revealed hypermethylation in at least 1 of the 4 analyzed genes, whereas all urine samples from normal controls were negative. The sensitivity of MSP assay for detecting E-cadherin, p16, p14 and RASSF1A in tumor cells in voided urine was 35%, 35%, 33%, and 65%, respectively. Diagnostic sensitivity was 75% for combining RASSF1A and p14, and 83% for RASSF1A, p14 and E-cadherin. Urine cytology, however, detect only 13 (28%) cases of cancer or suspicious cancer. For detecting superficial and invasive bladder tumor, urine cytology revealed a sensitivity of 23% (6/26) and 35% (7/20), respectively. In contrast, MSP detected hypermethylation in the urine of 80% (37/46) bladder cancer patients. Moreover, hypermethylation analysis of E-cadherin, p14 or RASSF1A genes in urine sediment DNA detected in 85% (22/26) of superficial, 85% (11/13) of low grade, 75% (15/20) of invasive and 79% (26/33) of high grade bladder cancers. Importantly, hypermethylation was detected in the urine DNA of 90% (18/20) superficial tumors with negative or atypia cytology.ConclusionsHypermethylation of E-cadherin, p14 or RASSF1A in urine sediment DNA is a potential biomarker for detecting superficial, low grade cancer. Besides, hypermethylation of these 3 genes is a valuable adjunct diagnostic marker to urine cytology, which can enhance the diagnostic accuracy and follow-up treatment of bladder cancer patients.     

尿脱落细胞CD44基因变异表达产物对膀胱癌诊断价值的研究

应用逆转录－聚合酶链反应和Ｓｏｕｔｈｅｒｎ印迹杂交技术对３０例尿液标本脱落细胞ＣＤ４４基因含Ｖ６外显子的表达产物进行检测，并与尿细胞检查结果进行比较。结果显示：９０％的膀胱癌尿脱落细胞均检测到ＣＤ４４基因含Ｖ６外显子的拼接变异转录物的过量表达；     

Immunocyt and the HA-HAase urine tests for the detection of bladder cancer: a side-by-side comparison

OBJECTIVES: The reliable detection of bladder cancer from urine specimen remains an unsolved problem. Especially superficial bladder cancer can be missed with urine tests. We assessed the sensitivity and specificity of the commercial Immunocyt test in a side-by-side comparison with the HA-HAase urine test and cytology. The Immunocyt test measures the immunocytological expression of sulfated mucin-glycoproteins and glycosylated forms of the carcinoembryonic antigen in urine. With the HA-HAase urine test the level of hyaluronic acid (HA) and its degrading enzyme hyaluronidase (HAase) are measured in an ELISA-like test. METHODS: A total of 94 consecutive patients were studied and among these 30 patients had bladder cancer and 64 were controls. Among bladder cancer patients, there were 14 pTa, 9 pT1, 5 pT2 and 2 carcinoma in situ (CIS) transitional cell carcinoma of the bladder, respectively. The controls consisted of 55 patients with a history of bladder cancer but no evidence of tumor at the follow-up cystoscopy and 9 benign prostatic hyperplasia (BPH) patients. The 30 transitional cell cancer specimens had 4 (13%) grade 1 tumors, 15 (50%) grade 2 tumors and 11 (37%) grade 3 tumors. Sensitivity and specificity as well as the positive and negative predictive values of each test were evaluated. RESULTS: The sensitivity of the HA-HAase urine test (83.3%; 25/30) was significantly higher than the Immunocyt at 63.3% (19/30) (p = 0.038, McNemar test) and cytology (73%; p < 0.05). The specificity of the HA-HAase test (78.1%; 50/64), Immunocyt (75%; 48/64) and cytology (79.7%; 51/64) were comparable. The prevalence of bladder cancer in our study was 31%. The positive predictive value (PPV) of the HA-HAase test (64.1%) was significantly higher than the Immunocyt test (54.3%). The negative predictive value (NPV) of the HA-HAase test (90.9%) was also higher than the Immunocyt test (81.3%). The PPV and NPV values for cytology were 62.9% and 86.4%, respectively. False negative patients in the HA-HAase urine test were 5 pTa tumors (2 G1, 2 G2 and 1 G3). False negative patients in the Immunocyt test were 7 pTa tumors (1 G1 and 6 G2), 3 pT1 (2 G2, 1 G3) and 1 pT2 G3, respectively. CONCLUSIONS: The sensitivity of the HA-HAase urine test is significantly higher than that of the Immunocyt test to detect bladder cancer. Specificity, as well as the PPV and NPV of the HA-HAase test were higher than that of the Immunocyt test. With a prevalence of 31% bladder cancer patients in all hematuria patients studied, a typical distribution of patients in a urological clinic is presented. Longer follow up of the study patients will give more information on the value of these tests in the detection of bladder cancer.     

检测膀胱癌患者尿液脱落细胞中Survivin的表达及意义

目的：探讨检测膀胱癌患者尿液脱落细胞中Survivin的表达及意义。方法：留取40例膀胱移行细胞癌患者、15例其他泌尿系统疾病患者和8例正常健康成人的新鲜尿液，离心收集脱落细胞，以逆转录多聚酶联反应(RT-PCR)检测膀胱癌患者尿液脱落细胞中Survivin的表达．并行尿脱落细胞学检查。结果：40例膀胱移行细胞癌患者尿脱落细胞中有38例检测出Survivin表达，而15例其他泌尿系统疾病患者和8例正常健康成人的尿脱落细胞中均未检测出Survivin的表达。以RT-PCR反应检测膀胱癌患者尿液脱落细胞中Survivin的方法敏感性为95％，特异性为100％。结论：初步的试验结果显示．RTPCR法检测膀胱癌患者尿液脱落细胞中Survivin的方法可以作为诊断膀胱癌的无创性方法。     

Use of the novel marker BLCA-1 for the detection of bladder cancer

PURPOSE: Bladder cancer specific nuclear structural alterations have been identified. We examined the expression pattern of one of these proteins, BLCA-1, in tissue and urine samples from individuals with bladder cancer as well as in samples from normal controls. MATERIALS AND METHODS: BLCA-1 sequence data were used to produce antibodies to this protein, which were used in immunoblot and enzyme-linked immunosorbent assays. RESULTS: BLCA-1 was detectable in tissue from patients with bladder cancer but not in normal adjacent areas of the bladder or in normal donor bladder tissue. This protein was also detectable in the urine of patients with bladder cancer by immunoblot and immunoassay. Using a cutoff of 0.025 optical density units (absorbance value) BLCA-1 was detected in 20 of 25 urine samples from patients with bladder cancer but in only 6 of 46 normal, high risk, prostate or renal cancer samples tested, resulting in a test with 80% sensitivity and 87% specificity. Expression of this protein did not appear to correlate with tumor grade. CONCLUSIONS: This research indicates that BLCA-1 is a urine based marker of bladder cancer which may be useful for the detection of this disease.     

尿脱落细胞CD44基因变异表达产物对膀胱癌诊断价值的研究

应用逆转录－聚合酶链反应和Ｓｏｕｔｈｅｒｎ印迹杂交技术对３０例尿液标本（１０例为膀胱癌，其中２例为复发性，２０例为非癌对照组）脱落细胞ＣＤ_（４４）基因含Ｖ_６外显于的变异表达产物进行检测，并与尿细胞学检查结果进行比较。结果显示：９０％（９／１０）的膀胱癌尿脱落细胞均检测到ＣＤ_（４４）基因含Ｖ_６外显子的拼接变异转录物的过量表达，而２０例非癌对照标本均未检测到这种变异表达产物。本技术诊断膀胱癌的敏感性为９０％（９／１０），较尿脱落细胞学检查的６０％（６／１０）明显提高，且无创伤，无痛苦。提示尿脱落细胞ＣＤ_（４４）基因变异表达产物检测是一种很有潜力的对膀胱癌早期诊断、监测复发和常规普查的简便实用的新方法。     

Genetic detection of bladder cancer by microsatellite analysis of p16, RB1 and p53 tumor suppressor genes

PURPOSE: We investigated the incidence of genetic alterations in urine specimens from patients with bladder cancer. MATERIALS AND METHODS: A total of 28 cytological urine specimens were assessed for microsatellite alternations, and 15 microsatellite markers were located on p53, RB1 and p16 regions. In 15 patients DNA from tumor specimens was also available. RESULTS: Loss of heterozygosity was detected in 26 of 28 patients (93%) in at least 1 microsatellite marker. Allelic losses were found in 18 patients (64%) for the p16 locus, in 8 (29%) for the RB1 locus and in 17 (61%) for the p53 region. In contrast, no microsatellite alterations were found in the normal group without evidence of bladder cancer. In 11 cases genetic alterations in the cytological urine specimens were not detectable in the corresponding tumor specimen, suggesting heterogeneity of bladder cancer. CONCLUSIONS: The detection of loss of heterozygosity in cytological urine specimens may be a prognostic indicator of early detection of bladder cancer. Our results suggest that microsatellite analysis of urine specimens represents a novel, potentially useful, noninvasive clinical tool to detect bladder cancer.