膀胱癌尿脱落细胞端粒酶活性检测及其临床意义

目的检测尿脱落细胞端粒酶活性并探讨其临床意义。方法应用改良的端粒重复序列扩增（ＴＲＡＰ）银染方法,分别对膀胱癌组织、正常膀胱组织,以及膀胱癌患者和非尿路上皮肿瘤患者的尿脱落细胞、膀胱冲洗液进行端粒酶活性检测。结果１２例正常膀胱组织均无端粒酶活性,４８例膀胱癌组织中４４例（９１．７％）端粒酶阳性。膀胱癌患者尿液及膀胱冲洗液中脱落细胞端粒酶阳性率分别为８３．３％（４０／４８）和８７．５％（４２／４８）。１２例分化良好（Ｇ１级）膀胱癌患者中,尿液和膀胱冲洗液中脱落细胞端粒酶阳性率分别为７５．０％（９／１２）和８３．３％（１０／１２）。结论尿脱落细胞端粒酶活性检测敏感性高,可用于膀胱癌的早期诊断和术后随访。     

尿脱落细胞端粒酶活性表达对尿路上皮癌的诊断价值

目的：探讨尿脱落细胞端粒酶活性表达对尿路上皮癌的诊断价值。方法：采用ＰＣＲ－ＣＬＩＳＡ法检测尿液中脱落的肿瘤细胞端粒酶活性,并以肾癌及非肿瘤患者的尿标本对照。结果：５９例尿路上皮癌患者尿脱落细胞端粒酶活性表达率为８８．１％,而肾癌患者为１０．０％,非肿瘤患者为２０．０％,前者与后两者比较,有极显著性差异（Ｐ〈０．０１）;不同分化程度及不同临床分期膀胱癌患者尿脱落细胞端粒酶活性表达率无显著性差异（Ｐ     

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。     

抑癌基因微卫星杂合性丢失在膀胱癌诊断中的应用

目的探讨抑癌基因p16、p53、VHL的微卫星（MS）DNA杂合性丢失（LOH）的发生率与膀胱移行细胞癌（TCC）临床分期、病理分级的关系。方法随机提取50例膀胱TCC组织标本DNA,并取50例癌旁正常膀胱黏膜作对照；提取40例患者尿液标本DNA,取位于p16、p53、VHL的6个微卫星位点引物行PCR扩增,8％变性聚丙烯酰胺电泳,银染结果检测杂合性丢失与TCC分期、分级的关系。结果50例TCC标本中,6个微卫星位点中至少1个位点LOH阳性者45例（90．0％）；40例尿液标本中,以上位点至少1个位点LOH阳性者35例（87．5％）,尿脱落细胞组织学检查阳性者25例（62．5％）,主要见于中、晚期肿瘤。p16、p53、VHL基因6个微卫星位点中,LOH阳性率由高到低分别为D9s259（76．3％）、D9s270（65．7％）、D17s786（50．0％）、D3s1284（41．7％）、D3s1038（41．2％）、D17s379（40．5％）。D9s259、D9s270、D3s1038、D17s786的LOH阳性率与TCC分期、分级无关,D3s1284、D17s379主要见于高分期、分级肿瘤。结论TCC组织标本和尿液标本抑癌基因微卫星位点LOH检测是一项新颖、高敏感的方法,D9s259、D9s270、D3s1038、D17s786可用于膀胱肿瘤的早期诊断,D3s1284、D17s379可作为膀胱肿瘤判断预后的指标。     

膀胱移行上皮癌患者尿及癌组织中Survivin表达的临床意义

目的：探讨Survivin对膀胱癌早期发现、常规筛选且无损伤的方法,研究其与肿瘤病理分级（期）的相关性。方法：留取53例膀胱移行上皮癌、20例泌尿系其他疾病、10例健康志愿者新鲜中段晨尿,同上53例术后癌组织。利用巢式RT—PCR、实时定量PCR技术检测Survivin的表达,同时行尿脱落细胞学及膀胱镜病检。结果：53例膀胱癌患者尿及癌组织中Survivin均有表达,敏感性为100％,特异性为90％。与尿脱落细胞学敏感性比较两者差异有统计学意义（P〈0．05）,与膀胱镜检比较差异无统计学意义。三种方法特异性比较差异无统计学意义（P〉0．05）。癌组织中Survivin的量与肿瘤病理分级（期）正相关（P〈0．01）。结论：检测尿脱落细胞中Survivin的表达有望成为临床诊断、筛检膀胱癌的较可靠方法。Survivin在膀胱的演进过程中可能起重要作用,可望作为检测膀胱癌恶化进展的指标。     

前列腺穿刺物端粒酶活性测定对前列腺癌的诊断价值

目的：通过检测前列腺穿刺物端粒酶活性，探讨其在前列腺癌诊断中的价值。方法：对41例可疑前列腺癌患者进行前列腺穿刺，对穿刺物行端粒酶活性测定及病理检查。端粒酶活性采用端粒重复放大程序（TRAP）PCR银染色法作定性检查，同时采用TRAP－PCR ELISA法作半定量测定。结果：26例前列腺癌患者中，端凿酶活性检测阳性22例，阳性率为84．6％，平均活性值为0．4506；15例非前列腺癌患者中，阳性4例，阳性率为26．7％，平均活性值为0．1263。两组之间端粒酶表达率及活性值差异均有显著性意义（P＜0．05或P＜0．01）；癌旁组织17例，阳性2例（11．8％）。前列腺癌端粒酶活性表达与肿瘤分级、分期及术前血清前列腺特异抗原无关（P＞0．05）。结论：前列腺穿刺物端粒酶活性测定是一种诊断前列腺癌的有用标志。     

端粒酶活性在人膀胱肿瘤组织中表达的临床意义

目的：探讨不同临床病理类型的膀胱肿瘤及癌旁组织端粒酶活性表达及临床意义，方法：以改良TRAP法测定91例膀胱肿瘤组织标本端粒酶活性表达。结果：83例膀胱移行细胞癌组织中78例检测到端粒酶活性，阳性率为94％，其对应的癌旁组织也有14％的检出率，8例膀胱乳头状瘤组织中4例检测到端粒酶活性，阳性率为50％，其对应的癌旁组织检出率为12％，端粒酶活性在不同临床病理类型的膀胱肿瘤及癌旁组织中表达差异无显著性意义（P＞0．05），结论：膀胱肿瘤及癌旁组织端粒酶活性的检测对肿瘤的诊断有重要意义。     

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。     

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。     

The clinical implications of telomerase activity in upper tract urothelial cancer and washings

OBJECTIVE: To measure telomerase activity in upper tract urothelial carcinomas (as renal pelvic tumours comprise nearly half of all kidney tumours in Taiwan, a much higher percentage than in other countries) and to determine whether telomerase activity could be used as an additional diagnostic marker in exfoliated cancer cells present in upper tract urothelial washing fluids, thus providing earlier diagnosis and treatment. Materials and methods Telomerase activity was assessed using the telomeric repeat amplification protocol assay in tissue samples from 31 upper tract urothelial carcinomas (from 29 patients). The feasibility of identifying cancer using telomerase activity in exfoliated cancer cells in 17 upper tract urothelial washing samples was also investigated. RESULTS: Telomerase activity was found in 30 (97%) of the 31 upper tract urothelial cancer tissue samples; telomerase activity was detectable in 95% of superficial cancers and in all 11 invasive tumours. The sensitivity of measuring telomerase activity was 100% for grade 1, 93% for grade 2 and 100% for grade 3 tumours. In contrast, telomerase activity was detected in only two (8%) of 26 normal adjacent tissue samples. When the telomerase activity of urothelial washing fluid was compared with that in the corresponding tumours, there was compatible telomerase activity in 15 of the 17 samples. Telomerase activity was more sensitive than voided urine cytology (15%) and washing fluid cytology (53%). In addition, the telomerase activity was high in metastatic lesions. CONCLUSION: Telomerase activity is present in most upper tract urothelial cancer tissues and may be present at an early stage of carcinogenesis. Telomerase activity can be detected in exfoliated cells in urothelial washing fluids in a high proportion of patients with upper tract urothelial cancer. These results suggest that measuring telomerase activity in the exfoliated cancer cells obtained from urothelial washing could be a potentially useful addition to the conventional diagnostic tools used to identify patients with upper tract urothelial carcinoma.     

Semi-quantitative analysis of telomerase activity of exfoliated cells in urine of patients with urothelial cancers: Causative factors affecting sensitivity and specificity

We previously reported that detection of telomerase activity in urinary exfoliated cells obtained from urothelial cancer patients by telomeric repeat amplification protocol (TRAP) assay is a more sensitive method of diagnosis than conventional urine cytologic examination, particularly in patients with grade 1 tumors. To establish this method as a noninvasive screening test for the diagnosis of urothelial cancers, we performed the semi-quantitative analysis of telomerase activity using Telomerase PCR ELISA?. Spontaneous voided urine samples were obtained from 65 urothelial and 58 non-urothelial cancer patients. When the mean + 2 standard deviation of telomerase activity in urine sediments of non-urothelial cancer patients was arbitrarily determined as a cut-off, the sensitivity of TRAP enzyme-linked immunosorbent assay (ELISA) and the conventional cytology were 57% and 35%, respectively. Detection rate was significantly higher in semi-quantitative TRAP assay than in conventional cytologic examination in grade 1 cancer patients (52% vs. 5%, p = 0.00195). False positives were detected in 5% of non-urothelial cancer patients without pyuria and in 11% of non-urothelial cancer patients with pyuria (p = 0.395). Telomerase activity was enhanced in some cases after phenol extraction or extracting epithelial cells by using Dynabeads of macroscopic hematuria and pyuria, indicating that hematuria and pyuria might contribute to false negatives. In conclusion, the TRAP-ELISA method is superior to the standard TRAP assay in quantitativeness and simplicity of the experimental procedure. Detection of telomerase activity in urine sediments is particularly useful for the diagnosis of low-grade tumors. However, telomerase activity in patients with high grade tumors often might be underestimated due to the excessive amount of exfoliated epithelia with necrotic tissues, hematuria, and pyuria.     

Telomerase activity in diagnosis of bladder cancer

OBJECTIVE: Telomerase is an enzyme that can reconstitute the ends of chromosomes after cell division and thus circumvent the damage that occurs in normal adult somatic cells during successive mitotic cycles. Immortal cells have short but stable chromosomes and increased telomerase activity. Transitional cell carcinoma (TCC) has only a few useful markers of diagnostic or prognostic importance. The objectives of this study were to determine whether there was a correlation between telomerase activities and the grade or stage of TCC and whether the activity of the enzyme could serve as a biochemical marker of this tumor. MATERIAL AND METHODS: Telomerase activity was determined by examining, using a polymerase chain reaction-based assay designed using the telomeric repeat amplification protocol (TRAP), urine cell pellets obtained from 42 bladder cancer patients, 18 patients with primary hematuria, 19 patients with benign urologic disease, 14 patients with urologic malignancies other than TCC and 20 healthy volunteers. RESULTS: Telomerase activity was found in 24/31 patients with bladder tumors (77.4% sensitivity) and in 5/77 patients without tumors (93.5% specificity). No correlation was found between telomerase activity and the grade or stage of the tumor. Although none of the urine cell pellets obtained from the 20 healthy volunteers demonstrated telomerase activity, positive telomerase activity was found in two subjects in the benign urologic disease group and in three subjects in the other urologic malignancy group. It was demonstrated that gross hematuria was the cause of false-negative results in six of the nine patients (66.7%). but washing the pellets four times and diluting them before the TRAP assay solved this problem. CONCLUSION: These results indicate that telomerase activity may be a promising marker for TCC but the technical aspects of the technique must be improved before it is used in routine clinical practice as a standard method. False-negative results obtained using gross hematuric urine should be carefully reevaluated and cell pellets should be washed again and diluted before analysis.     

Telomerase activity Simplification of assay and detection in bladder tumor and urinary exfoliated cells

Detection of telomerase activity can differentiate malignant from benign cells. However, the original telomeric repeat amplification protocol (TRAP) methods had a number of limitations including a radioisotope labeling [α(32)P] dCTP [α(32)P] dGTP system. We developed digoxigenin labeled CX primer to detect telomerase activity without using radioisotope and attempted to detect telomerase activity of bladder tumor and exfoliated cells in bladder cancer patients. Telomerase activity was detected in 5 (71%) of 7 patients diagnosed with grade 1, 31 (97%) of 32 grade 2, and 11 (100%) of 11 grade 3 bladder tumors. In urinary exfoliated cells, 32 (82%) of 39 grades 1 or 2 bladder tumors were positive for telomerase activity but 20 (51%) of 39 were positive for urinary cytology (P < 0.01). Ten (91%) of 11 of grade 3 tumors were positive for telomerase activity and 11 (100%) of 11 were positive urinary cytology. Three of 100 noncancerous patients were positive for telomerase activity. Sensitivity, specificity, and positive predictive value of telomerase activity assay in urinary exfoliated cells were 84%, 97%, and 93%, respectively. Telomerase activity may be a useful diagnostic marker to detect the existence of immortal cancer cells in the urine.     

Evidence for elevated telomerase activity in small cell carcinoma of the bladder

Telomerase activity was found to be elevated using a quantitative assay on snap-frozen protein extracts of exfoliated cells in urine and bladder washings and tumor tissue obtained from a male patient with small cell carcinoma of the bladder. To the best of our knowledge, this is the first demonstration of elevated values of telomerase activity in genitourinary small cell carcinoma and is in keeping with the findings in primary lung locations.     

Telomerase activity is correlated with lower grade and lower stage bladder carcinomas

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that compensates for the progressive erosion of telomeres. The increasing interest in telomerase is motivated by the demonstration that most human carcinomas are telomerase positive. The potential use of telomerase activity in bladder carcinomas using a urine sample has been reported in several studies. However, little is known about the detection of telomerase activity in bladder carcinoma tissues. Herein, we investigate telomerase activity in bladder carcinoma tissues according to grade (G) and stage. MATERIAL AND METHODS: Telomerase activity was assayed by polymerase chain reaction enzyme-linked immunosorbent assay methods. Malignant lesions were assessed in 37 patients with bladder carcinoma and no malignant lesions were assessed in five patients with dysplasia or inflammatory bladder lesions. RESULTS: Twenty-three out of 37 carcinoma samples were telomerase-positive and one out of five control samples without carcinoma was telomerase-positive. The positive rates according to stage and grade were 83.3% for superficial and 42.1% for invasive stages and 83.3% for G1, 66.7% for G2 and 40.0% for G3. Telomerase activity was correlated with lower grade and lower stage bladder carcinomas. CONCLUSION: These results strongly suggest that reactivation of telomerase may differ between superficial and invasive bladder carcinomas and also between low grade and high grade bladder carcinomas.     

The molecular detection of circulating tumor cells in bladder cancer using telomerase activity.

PURPOSE: The detection of circulating tumor cells and micrometastases may have important prognostic and therapeutic implications. We investigated telomerase activity as a molecular marker for detecting bladder carcinoma cells in blood. MATERIALS AND METHODS: Peripheral blood mononuclear cells were isolated from whole blood using Ficoll/Hypaque. Immuno-magnetic beads labeled with an epithelial specific antibody were used to harvest epithelial cells from peripheral blood mononuclear cells. Telomerase activity was detected in this select population using the telomerase-polymerase chain reaction-enzyme-linked immunosorbent assay test based on the telomerase repeat amplification protocol method. The clinical applicability of this technique was explored by evaluating 30 patients with muscle invasive or metastatic bladder carcinoma and 17 healthy volunteers. RESULTS: Telomerase expression was detected in 27 of the 30 patients (90%) with high grade, muscle invasive or metastatic bladder cancer but in none of the 17 healthy controls. CONCLUSIONS: This test is a minimally invasive and specific approach for detecting circulating epithelial cells in patients with bladder cancer. This method may have great value for monitoring cancer progression.     

膀胱癌组织端粒酶活性的研究

目的　探讨膀胱癌组织端粒酶活性的临床意义。　方法　应用银染端粒重复序列扩增法检测 42例膀胱癌及其癌旁组织的端粒酶活性。　结果　 3例正常膀胱组织端粒酶表达均阴性 ;42例膀胱癌组织中端粒酶表达阳性 35例 (83 .3 % ) ,癌旁组织端粒酶表达阳性 7例 (16 .7% ) ;浸润癌或有淋巴结或远处转移者端粒酶表达阳性率高于非浸润癌或无转移者 ,但差别无显著性意义 (P >0 .0 5 )。　结论　端粒酶是膀胱癌较理想的肿瘤标记物之一。