Urine: a suitable sample for deoxyribonucleic acid flow cytometry studies in patients with bladder cancer

Deoxyribonucleic acid flow cytometry of bladder washings has proved to be a valuable procedure for the diagnosis and followup of patients with transitional cell carcinoma. However, for this procedure to gain maximal acceptance, it should be possible to use voided urine specimens instead of bladder washings. To evaluate this possibility we compared histogram findings for 114 bladder washings and 122 concomitantly obtained urine samples (voided and catheterized) from 89 consecutive patients who had active or a history of transitional cell carcinoma. Unsatisfactory histograms were obtained in 4 per cent of the urine samples and in 2.6 per cent of the bladder washing samples. The satisfactory rate for voided or catheterized urine samples was the same. We conclude that in this patient population satisfactory deoxyribonucleic acid histograms can be obtained from samples of voided urine.     

A comparative study of the sensitivity of bladder washing flow cytometry, voided urine cytology and bladder washing cytology in the detection of bladder carcinoma

In order to evaluate flow cytometric deoxyribonucleic acid measurement (FCM) of bladder washing in the diagnosis of bladder carcinoma, the sensitivity of voided urine cytology, bladder washing cytology and bladder washing FCM was tested in 76 samples from 56 patients with histologically proven bladder carcinomas. The positive rates were 43.2% and 75.7% in bladder washing cytology and bladder washing FCM, respectively. On the other hand, 36.5% and 57.1% positive rates for once- and three-times-voided urine cytology, respectively, were obtained. Bladder washing cytology and bladder washing FCM were positive in 20% and 70% patients with a histological diagnosis of atypia or dysplasia, respectively. The sensitivity of bladder washing FCM according to the tumor grade was 33.3%, 81.9% and 88.9% for grade-1 (G-1), G-2 and G-3 tumors, respectively. The sensitivity of bladder washing cytology according to the tumor grade was 0, 40.9% and 77.8% for G-1, G-2 and G-3 tumors, respectively. The sensitivity of three-times-voided urine cytology was 25.0%, 55.6% and 83.3% for G-1, G-2 and G-3 bladder tumors, respectively, and it was superior to that of single bladder washing cytology. These results indicate that FCM is more sensitive than voided urine cytology and/or bladder washing cytology in patients with bladder carcinoma. FCM may indicate urothelial neoplasia before it is apparent on urine cytology, especially against a background of inflammation. Therefore, FCM is valuable for case finding in suspect populations or for follow-up cases with diagnosed bladder cancer.     

Fractionated urinary cytology in the follow-up of bladder cancer

Cytological examination of voided urine is an established investigation in urological practice. In a pilot study of 50 patients with histologically proven transitional cell carcinoma of the bladder, urine cytology was undertaken on samples from the initial, mid-stream and terminal parts of the void. Analysis showed that although the cell density varied between the samples in some cases, no part of the void was consistently richer in benign or malignant cells and similar cell types were seen in every sample from any given patient. It was concluded that fractionated cytology did not improve the diagnostic accuracy of urinary cytology and that any sample of urine was suitable for cytological purposes.     

膀胱癌患者尿沉渣中微卫星不稳定性的临床意义

目的 探讨微卫星不稳定性（ＭＳ）在膀胱癌中的表现及其临床诊断价值。方法 利用ＰＣＲ方法检测了３５例膀胱癌患者术前尿沉渣中１３个微降临位点,并分析了微降临不稳定性（ＭＳＩ）及杂合性丢失（ＬＯＨ）。对其中２５例患者进行了随访。结果 ３５例膀胱癌经尿沉渣微降临分析,３１例（８８．６％）可检出膀胱癌。１２例复发肿瘤中１０例可见微卫星改变（８３％）,３例首先发现尿沉渣微卫星改变,经１～６个月继续随一,经膀胱     

Flow cytometric study comparing paired bladder washing and voided urine for bladder cancer detection

Paired bladder washings and voided urines from bladder cancer patients were compared as sources of exfoliated cells for detection of bladder carcinoma by flow cytometry (FCM). Bladder specimens fixed in 25% ethanol within sixty minutes of collection were found to be superior to unfixed bladder specimens. The percentage of specimens with good DNA resolution was greater for bladder washings (67% unfixed, 90% fixed) than voided urines (41% unfixed, 66% fixed). There was no difference in DNA resolution between specimens that remained unfixed less than one day, one day, or two days suggesting that the cells undergo the majority of degradation within a critical period soon after collection. Once fixed, there was no difference in DNA resolution for up to nineteen days, which suggests the feasibility of specimen transport to central FCM laboratories. Eighteen percent of unfixed bladder washings and 33 percent of unfixed voided urine specimens contained an insufficient number of cells (less than 5,000) at the time of analysis compared with 6 percent bladder washings and 17 percent voided urines fixed in 25% ethanol. Flow cytometry and cytology results were concordant in 28/43 (65%) of fixed bladder washings and 9/13 (69%) of fixed voided urine. Voided urine was unreliable in providing consistent FCM data due to the high number of specimens with poor resolution or insufficient cells and is not recommended as a substitute for bladder washing when screening high-risk populations or monitoring patients with past history of bladder cancer.     

Flow cytometry: role in monitoring transitional cell carcinoma of bladder

The ability to detect and monitor the course of transitional cell carcinoma (TCC) of the bladder using DNA histograms obtained from flow cytometry was studied. Voided urine and barbotage specimens were collected from patients with active TCC or a past history of TCC. These specimens were submitted to routine cytologic and flow cytometric analyses. Samples were considered to be positive if they met one or more of three criteria: if they had aneuploid or tetraploid peaks, if the DNA index of the major G1 peak was shifted more than 10 per cent from that of diploid cells, or if 15 per cent or more of the cells fell to the right of the major diploid G1 cell population thereby constituting a significant hyperdiploid cell population. Using these methods for patients with active disease, the detection rate was 91 per cent. In patients with a past history of TCC, positive histograms preceded the appearance of visible tumor in one third of the cases. Flow cytometry proved to be an excellent way of following patients with a past history of TCC or of screening patients suspected of having active disease. Following this protocol, few biologically active tumors go undetected. However, in 112 patients without a history of bladder cancer, the false positive or suspicious rate was 38 per cent. Before flow cytometry can be recommended as a widespread screening method for patients thought to be at risk of TCC of the bladder developing, this suspicious group will have to be eliminated.     

Initial evaluation of the diagnostic performance of the new urinary bladder cancer antigen test as a tumor marker for transitional cell carcinoma of the bladder

PURPOSE: We evaluated the diagnostic performance of the new noninvasive bladder cancer test on voided urine samples from patients with transitional cell carcinoma compared to symptomatic and asymptomatic controls. MATERIALS AND METHODS: Urinary bladder cancer antigen was measured in urine from 86 patients with active transitional cell carcinoma of the bladder (group 1), 76 patients free of transitional cell carcinoma as confirmed by cystoscopy at followup (group 2), 25 patients with other benign urological diseases (group 3), 25 patients with other malignant pathological conditions (group 4) and 30 healthy subjects free of urological diseases (group 5). RESULTS: Mean urinary bladder cancer antigen concentrations were 104.84, 4.57, 11.79, 48.87 and 1.38 microg/l, for groups 1 to 5, respectively, which was statistically different (p = 0.00005) except for groups 1 and 4 (p = 0.187). Sensitivity was 87.0% (95% confidence interval 79.2 to 92.7) and specificity was 86.8% (77.1 to 93.5%), and both were optimized by receiver operating characteristics plot analysis at a threshold value of 9.74 microg/l using asymptomatic (group 2) compared to known cancer (group 1) cases. CONCLUSIONS: Urinary bladder cancer antigen might have a role as a potential tumor marker for diagnosing transitional cell carcinoma of the bladder.     

全反式维甲酸诱导膀胱癌Ｔ２４细胞株凋亡的研究

目的 :探讨全反式维甲酸 (ATRA)对人膀胱癌 T2 4细胞株的凋亡诱导作用。方法 :应用荧光显微镜、流式细胞仪及 DNA琼脂糖凝胶电泳等技术研究 T2 4细胞凋亡。结果 :用 3× 10 - 6 m ol/ L 的 ATRA处理 T2 4细胞6天 ,荧光显微镜下计数 T2 4细胞凋亡率为 15 .2 0 % ,对照组为 0 .0 1% (P <0 .0 5 ) ;流式细胞仪测定 T2 4细胞凋亡率为 15 .31% ,对照组为 1.49% ;T2 4细胞 DNA琼脂糖凝胶电泳呈现凋亡特征性梯形条带。结论 :ATRA对人膀胱癌 T2 4细胞株有凋亡诱导作用 ,为 ATRA应用于膀胱癌临床治疗提供了实验依据     

核基质蛋白22检测在膀胱癌筛查中的应用

目的：探讨检测核基质蛋白22(NMP22)在膀胱癌筛查中的应用价值。方法：检测129例血尿患者的尿NMP22，并分为膀胱癌组和非肿瘤组进行比较。结果：膀胱癌组患者尿NMP22值高于非肿瘤组(P＜0．01)。结论：尿NMP22检测具有较高的敏感性和特异性，在膀胱癌筛查中可以选择应用。     

粘蛋白7在膀胱癌尿脱落细胞中的表达及意义

目的:探讨粘蛋白7(mucin7,MUC7)在膀胱移行细胞癌(TCC)诊断中的意义。方法:采用巢式逆转录聚合酶链反应(nested RT-PCR)方法检测MUC7mRNA在TCC患者尿脱落细胞中的表达。结果:68例TCC患者中有42例尿液MUC7表达阳性,敏感性为62%;MUC7表达与临床分期有关而与病理分级无关。对照组25例中有3例在尿液中MUC7为阳性表达,特异性为88%。结论:用RT-PCR的方法检测尿脱落细胞中的MUC7,具有较高的膀胱癌诊断价值。     

Detection of onco-fetal bladder antigen in urine of patients with transitional cell carcinoma

Studies of antigens associated with transitional cell carcinoma were extended by using murine IgM monoclonal antibody E7, developed earlier by this laboratory. These antibodies react preferentially with human bladder tumors and transitional cell carcinoma (TCC) cell line 647V. We now report that monoclonal antibody E7 detected the presence of antigen in midgestational and third trimester amniotic fluids, and in urine of patients with advanced transitional cell carcinoma. Western blot analysis showed that the antigen present in amniotic fluids consists of a sharp band with molecular weight greater than 200 kdaltons. A similar molecular weight pattern was seen with the solubilized membrane of 647V. A sensitive and convenient sandwich ELISA was developed and the urine of patients with bladder cancer was assayed for the presence of the E7 antigen. Antigen was detected in the urine of patients with advanced transitional cell carcinoma but not in the urine of normal adults or in urine from patients with prostate cancer, renal cell carcinoma, or benign prostate hyperplasia. An inhibition enzyme immunoassay was developed with monomeric forms of the E7 antibody and confirmed the presence of antigen in the urine of patients with TCC. We conclude that the E7 antigen is an onco-fetal antigen expressed in patients with transitional cell carcinoma of the bladder.     

Multitarget fluorescence in situ hybridization assay detects transitional cell carcinoma in the majority of patients with bladder cancer and atypical or negative urine cytology

PURPOSE: The multitarget fluorescence in situ hybridization (FISH) probe set UroVysion (Vysis, Downers Grove, Illinois), containing probes to chromosomes 3, 7 and 17, and to the 9p21 band, has been recently shown to have high sensitivity and specificity for detecting transitional cell carcinoma. In this study we retrospectively tested 120 urine samples from patients with atypical, suspicious and negative cytology for whom concurrent and followup bladder biopsy data were available. We evaluated the ability of FISH to identify malignant cells in cytologically equivocal or negative cases. MATERIALS AND METHODS: Archived slides from 120 voided (47) or instrumented (73) urine cytology specimens from patients with concurrent bladder biopsy and a minimum of 12 months of biopsy followup were subjected to hybridization with UroVysion. The cohort included patients with biopsy proven transitional cell carcinoma, which was grades 1 to 3 in 23, 35 and 24, respectively, and stages pTis in 3, pTa in 64, pT1 in 6, pT2 in 6 and pT4 in 3, while it showed negative histology in 38. Cytology findings were suspicious, atypical and negative for transitional cell carcinoma in 31, 49 and 40 cases, respectively. A positive FISH result was defined as 5 transitional cells or greater with a gain of 2 or more of chromosomes 3, 7 or 17, 12 cells or greater with 9p21 deletion, or 10% or greater of cells with isolated trisomy of 1 of chromosomes 3, 7 and 17. RESULTS: All except 12 of the 82 biopsy proven transitional cell carcinoma cases (11 pTa and 1 pT1 tumors) were positive by FISH (85% sensitivity). Sensitivity in patients with suspicious, atypical and negative cytology was 100%, 89% and 60%, respectively. Nine patients with atypical cytology had positive FISH in the setting of a negative concurrent bladder biopsy. However, 8 of these 9 patients (89%) had biopsy proven transitional cell carcinoma within 12 months following the date when the sample tested by FISH was obtained. The last of these patients with false-positive results had previously documented pTis disease, which was also present in the next bladder biopsy 15 months following the positive FISH result. The remaining 29 specimens from patients with negative biopsy and a negative 12-month followup tested negative by FISH (97% overall specificity). CONCLUSIONS: The UroVysion FISH assay provides high sensitivity and specificity to detect transitional cell carcinoma in cytologically equivocal and negative urine samples. These results emphasize the important role of this assay in the management of bladder cancer.     

Transitional cell cytokeratins as a second parameter in flow cytometry of bladder cancer

Summary Since the detection of abnormal DNA content in the cells of transitional cell carcinoma can be obscured by the presence in the population of diploid stromal and inflammatory cells, it would be desirable to have a second flow cytometric parameter to complement DNA analysis. The specific identification of carcinoma cells using monoclonal antibodies directed against epithelial intermediate filament proteins (cytokeratins) would provide a method with which to identify the transitional cells in solid tumors, voided urines or bladder washings. As a preliminary study we have tested cell-free urine samples using a double antibody radiometric assay (IRMAK) for cytokeratins released from transitional cells. The additional information from cytokeratin analyses increased the specificity of disease detection.Supported in part by grants from Triton Biosciences, Inc., and by 1V01 CA41034-01, NCI, DHSS     

PIWIL2在人膀胱移行细胞癌肿瘤干细胞样细胞中的表达及意义

目的 研究PIWIL2在人膀胱癌肿瘤干细胞样细胞(CSLC)中的表达和意义,探讨PIWIL2在靶向肿瘤干细胞治疗中的作用.方法 用无血清悬浮培养法从人膀胱移行细胞癌细胞株BIU-87中分离获得悬浮细胞球,流式细胞仪检测细胞表面分子标志CD133和CD44的表达,免疫磁珠分选系统分离CD133+CD44+细胞;分别采用T...     

The effect of antisense Bcl-2 oligonucleotides on Bcl-2 protein expression and apoptosis in human bladder transitional cell carcinoma

PURPOSE: Bcl-2 is an important determinant of transitional cell carcinoma of the bladder recurrence and progression as well as a factor in patient response to chemotherapy or radiotherapy. We determined Bcl-2 down-regulation after antisense oligonucleotide therapy and synergism with mitomycin C in transitional cell carcinoma of the bladder. MATERIALS AND METHODS: Bcl-2 protein was quantified using flow cytometry and immunohistochemistry in 4 bladder cancer cell lines, in bladder washings from 6 patients with carcinoma in situ and in 16 patient tumor samples. The synergistic effects of antisense oligonucleotides G3139 and 2009, and mitomycin C were investigated in 4 cell lines, while 2009 down-regulation was examined in 20 tumor explants in an ex vivo model. RESULTS: Bcl-2 protein expression was found in all 4 cell lines and in 5 of the 6 cell populations derived from patients with carcinoma in situ. Of the 16 tumors 7 were classified positive by frozen section immunohistochemistry and quantitative flow cytometry. G3139 and 2009 down-regulated Bcl-2 protein expression in all 4 cell lines and 2009 down-regulated Bcl-2 protein expression in half of the Bcl-2 positive tumor specimens. There was only evidence in 1 cell line, T24/83, that Bcl-2 protein expression down-regulation enhanced mitomycin C induced apoptotic cell death. CONCLUSIONS: Bcl-2 was expressed in a significant proportion of bladder tumors and in carcinoma in situ. Therefore, antisense oligonucleotides represent a viable strategy for Bcl-2 protein down-regulation. However, it may not always translate into an increased level of mitomycin C induced apoptosis in transitional cell carcinoma of the bladder.     

Monoclonal antibodies against transitional cell carcinoma for detection of malignant urothelial cells in bladder washing

The diagnosis of transitional cell carcinoma by cytological examination of exfoliated urinary cells is important in the early detection and followup of patients with this disease. Proper interpretation requires a skilled pathologist. Accuracy also is influenced by collection methods and nonmalignant pathological conditions of the bladder. An immunocytochemical technique using monoclonal antibodies G4 and E7 successfully identified tumor-associated antigens on the surface of transitional carcinoma cells obtained by bladder washings. The method, which uses immunoperoxidase staining, was compared to conventional Papanicolaou staining of bladder washings from 75 patients with and without transitional cell carcinoma. Patients were divided into 4 groups: group 1 (nontumor control)--15 patients with no pathological condition of the bladder or nonmalignant urological diseases, group 2 (nontransitional cell carcinoma)--19 patients with other urological malignancies, group 3-18 patients with active transitional cell carcinoma and group 4-23 patients with a history of transitional cell carcinoma but no evidence of tumor at the time of the washing. The incidence of positive staining in these groups was 0, 5, 78 and 0 per cent, respectively. The diagnostic value of immunoperoxidase staining was similar to that of Papanicolaou staining in the control group and in patients with high grade transitional cell carcinoma, and provided specific morphological criteria not possible by conventional cytology studies. Interpretation of immunoperoxidase staining was difficult in washings with a large number of inflammatory cells if endogenous peroxidase activity was not blocked properly. The application of the immunoperoxidase staining method for diagnosis of low grade tumor is under further investigation.     

膀胱肿瘤患者红细胞免疫功能的变化

目的 探讨膀胱肿瘤患者的红细胞免疫功能状态及与Ｔ淋巴细胞亚群改变的关系。方法 测定３４例膀胱移行细胞癌患者的红细胞Ⅰ型补体受体花环率（Ｃ３ｂＲＲ）、免疫复合物花环率（ＩＣＲ）及４项肿瘤红细胞花环率［直向肿瘤红细胞花环试验（ＤＴＥＲ）、促肿瘤红细胞花环试验（ＥＴＥＲ）、协同肿瘤红细胞花环试验（ＡＴＥＲ）及自然肿瘤红细胞花环试验（ＮＴＥＲ）］,同时用流式细胞仪（ＥＴＥＲ）检测Ｔ细胞亚群,与年龄相近的３     

Non-invasive diagnostic tests for bladder cancer: a review of the literature

Transitional cell carcinoma of the bladder is the second most common malignancy of the genitourinary tract. Cystoscopy and urine cytology are the traditional most used techniques for diagnosis and surveillance of superficial bladder cancer. Urine cytology is specific for diagnosis of bladder cancer but sensitivity results not high, particularly in low-grade disease. Voided urine can be easily obtained and therefore additional diagnostic urine tests would be ideal for screening or follow-up of transitional cell carcinoma. A number of studies have focused on the evaluation of urinary markers that hold promise as non-invasive adjuncts to conventional diagnostic or surveillance techniques. In this review we discuss several new urinary markers (test for bladder tumor antigen, NMP22, fibrin degradation products, telomerase, fluorescence in situ hybridization test, flow cytometry) and their role in detection and follow-up of bladder cancer. Most of these markers have higher sensitivity than urine cytology, but voided urine cytology has the highest specificity.     

Upper urinary tract transitional cell carcinoma after total cystectomy for bladder cancer

During a 17-year-period from 1967 to 1983, 110 total cystectomies for transitional cell bladder cancer have been performed in our clinic. During the postcystectomy period, upper urinary tract urothelial cancer developed in seven patients (6.4%). In every case a multifocal, low stage transitional cell cancer had been found in the bladder. The time between the cystectomy and discovery of the upper tract tumour varied from less than three months to almost 13 years. In five cases the first sign of occurrence of the tumour was malignant conduit urine cytology, in two macroscopic haematuria with subsequent malignant cells in urine. In one patient bilateral renal pelvic tumours were found. Five patients could be surgically treated. The need for regular conduit urine cytological studies at short intervals in patients with multifocal low stage and high grade transitional cell carcinoma in the cystectomy specimen is emphasised.     

Flow cytometric analysis of primary and metastatic bladder cancer

A total of 22 patients with high grade P2-4N+ transitional cell carcinoma of the bladder underwent flow cytometric analysis of nuclei obtained from paraffin embedded specimens from the primary (bladder) and metastatic (lymph node) sites. Tumor heterogeneity was defined as polyclonal aneuploidy of the primary tumor (not identified in the population studied) or as a difference in the deoxyribonucleic acid index of the primary and metastatic sites of 0.20 or more (8 patients). With these criteria 8 patients (36%) had heterogeneous tumors and 14 (64%) had homogeneous tumors. The median survival of 14 patients with aneuploid and 8 with diploid primary tumors was 17.5 and 8.0 months, respectively (p equals 0.08, Lee-Desu test). When patient survival was compared to the ploidy of the metastatic site, or in patients with diploid primary and metastatic lesions versus deoxyribonucleic acid aneuploidy at either the primary and/or metastatic site, the aneuploid tumors had a longer survival but this difference was not significant (p equals 0.13 and 0.23, respectively). Our study demonstrates the value of flow cytometry to identify primary metastatic tumor heterogeneity. It also suggests that the presence of metastasis may be a more important factor to define the biological potential of transitional cell carcinoma than is deoxyribonucleic acid ploidy.