Flow cytometry deoxyribonucleic acid determinations and cytology of bladder washings: practical experience

Flow cytometry deoxyribonucleic acid measurements of bladder washings, routine bladder irrigation cytology and cystoscopic results were compared in a prospective fashion during 33 months of clinical urology practice. Of the 204 patients (286 specimens) studied 74 had bladder tumors or a history of bladder tumors and 130 had other genitourinary pathological conditions, including hematuria, cystitis, benign prostatic hypertrophy or prostatic carcinoma. Flow cytometry and cytology agreed with the cystoscopic findings in 93 per cent of the cases, whereas flow cytometry agreed with cytology in 84.6 per cent. When combined, flow cytometry and cytology were negative in only 1 case when several papillomas or grade I papillary carcinomas were present on cystoscopy, yielding a diagnostic accuracy of 99 per cent. The false negative rates for flow cytometry and cytology were 4.3 and 5.2 per cent, respectively, with false positive rates of 4.2 and 3.1 per cent, respectively. As expected the specimens that were missed by cytology were low grade tumors and those missed by flow cytometry were usually ulcerated invasive tumors. We believe that flow cytometry is a valuable adjunct to cytology and cystoscopy in the diagnosis, management and followup of patients with known or suspected bladder cancer. In addition, criteria for the diagnosis of cancer are changed such that specimens with a deoxyribonucleic acid tail and a large amount of hyperdiploid cells without a distinct stem line are considered suspicious and indicative of bladder pathology but they are not synonymous with the presence of carcinoma.     

Flow cytometric analysis of primary adenocarcinoma of the bladder

The flow cytometric findings of bladder irrigation specimens from 4 patients with histologically confirmed primary adenocarcinoma of the bladder are reported. Cells in specimens from 3 patients showed deoxyribonucleic acid aneuploidy and a simultaneous preoperative urinary cytology study was positive. One patient had normal flow cytometry studies, whereas preoperative cytology results were suspicious. These findings indicate that at least some, perhaps most, primary adenocarcinomas of the bladder have abnormalities of deoxyribonucleic acid content. Flow cytometry should be of value in detecting and monitoring these cases, as it is for epidermoid (urothelial) carcinomas.     

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.     

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.     

Flow cytometry of ethanol-fixed versus fresh bladder barbotage specimens

Saline bladder barbotage specimens were obtained from 99 patients for comparison of flow cytometric deoxyribonucleic acid analyses using fresh versus ethanol-fixed cell preparation techniques. The analyses were consistent for the 2 methods in 87 (88 per cent) of the 99 cases (p less than 0.0001, Kappa statistic for agreement). Since accurate, reproducible results are obtainable on fixed bladder washings, samples may be sent to flow cytometry centers for analysis.     

Cancer detection by quantitative fluorescence image analysis

Quantitative fluorescence image analysis is a rapidly evolving biophysical cytochemical technology with the potential for multiple clinical and basic research applications. We report the application of this technique for bladder cancer detection and discuss its potential usefulness as an adjunct to methods used currently by urologists for the diagnosis and management of bladder cancer. Quantitative fluorescence image analysis is a cytological method that incorporates 2 diagnostic techniques, quantitation of nuclear deoxyribonucleic acid and morphometric analysis, in a single semiautomated system to facilitate the identification of rare events, that is individual cancer cells. When compared to routine cytopathology for detection of bladder cancer in symptomatic patients, quantitative fluorescence image analysis demonstrated greater sensitivity (76 versus 33 per cent) for the detection of low grade transitional cell carcinoma. The specificity of quantitative fluorescence image analysis in a small control group was 94 per cent and with the manual method for quantitation of absolute nuclear fluorescence intensity in the screening of high risk asymptomatic subjects the specificity was 96.7 per cent. The more familiar flow cytometry is another fluorescence technique for measurement of nuclear deoxyribonucleic acid. However, rather than identifying individual cancer cells, flow cytometry identifies cellular pattern distributions, that is the ratio of normal to abnormal cells. Numerous studies by others have shown that flow cytometry is a sensitive method to monitor patients with diagnosed urological disease. Based upon results in separate quantitative fluorescence image analysis and flow cytometry studies, it appears that these 2 fluorescence techniques may be complementary tools for urological screening, diagnosis and management, and that they also may be useful separately or in combination to elucidate the oncogenic process, determine the biological potential of tumors and monitor the results of chemopreventive, immunological and chemotherapeutic regimens. To our knowledge there has been no study in which quantitative fluorescence image analysis and flow cytometry were compared directly to assess the relative strengths and weaknesses for urinary tract cytology. Such a study could provide important information for urologists.     

Positive urinary cytology after tumor resection: an indicator for concomitant carcinoma in situ

Concomitant urothelial atypia (grade II atypia or carcinoma in situ) is predictive of new tumor growth after transurethral tumor resection. Concomitant urothelial atypia can be demonstrated by pre-selected site mucosal biopsies. However, a number of patients have new tumors despite normal pre-selected site biopsies. To investigate whether urinary cytology is a better indicator for concomitant urothelial atypia than pre-selected site biopsies, we studied in bladder tumor patients the correlation between the findings of pre-selected site biopsies (8 per patient) at tumor resection and urinary cytology (2 per patient) after successful resection. Concomitant urothelial atypia was demonstrated by biopsies in 52 per cent of the patients, of whom 60 per cent had grade II atypia and 40 per cent had carcinoma in situ. All patients with concomitant carcinoma in situ in biopsies had positive cytology findings. Of the patients with concomitant grade II atypia in biopsies 15 per cent had negative cytology studies. In 48 per cent of the patients no urothelial atypia in pre-selected site biopsies was demonstrable. However, cytology was positive, that is neoplastic cells were present, in 64 per cent of these specimens (19 patients). Of the 19 patients 16 currently have had demonstrable urothelial atypia in pre-selected site mucosal biopsies at a later occasion. We conclude that urinary cytology seems to be a better indicator for the presence of concomitant urothelial atypia than pre-selected site mucosal biopsies and, therefore, it can be used as a screening procedure for patients without demonstrable concomitant carcinoma in situ at tumor resection.     

Correlation of histopathological, cytological and flow cytometric findings in neoplastic and nonneoplastic lesions of the bladder

Biopsy diagnosis, urine cytology and flow cytometry features of urine and bladder washings were compared in 81 cases of benign and malignant bladder disease. Some patients were followed by urine cytology and flow cytometry during treatment of tumors. There was a good correlation of the 3 parameters in relatively high grade tumors (II and III) but an inconsistent appearance of cells in the urine diagnosable by either urine cytology or flow cytometry for low grade lesions. High grade flat neoplasms may be detectable by urine cytology and flow cytometry before they are visible on endoscopy. Flow cytometry may indicate urothelial proliferation before it is apparent on urine cytology. Exfoliative benign lesions are diagnosed readily by the combination of urine cytology and flow cytometry. In benign and malignant lesions shedding of viable cells and, therefore, accessibility to urine cytology and flow cytometry are inconsistent. The need for repeated examinations in the case of low grade lesions suggests that the method is not reliable for single test screening programs but it is a valuable means of followup in cases of suspected or diagnosed disease.     

Flow cytometry as a predictor of response and progression in patients with superficial bladder cancer treated with bacillus Calmette-Guerin

Simultaneous bladder wash flow cytometry, voided urinary cytology and cystoscopic examinations were performed at 3-month intervals during a median of 18 months (range 5.5 to 50 months) in 65 patients receiving intravesical bacillus Calmette-Guerin treatment for superficial bladder cancer. Of the 65 patients treated 36 (56 per cent) had a complete response, 12 (18 per cent) had no response and 17 (26 per cent) had progression. Results of examinations at 6 months suggested that a negative bladder wash flow cytometry (29 of 36 patients, r equals 0.73, p less than 10(-7) is a strong predictor of response to bacillus Calmette-Guerin, comparable with cytological (r equals 0.60, p less than 10(-7) or cystoscopic (r equals 0.38, p less than 0.005) examinations alone or combined with cytology (r equals 0.74, p less than 10(-7)). At 6 months a positive bladder wash flow cytometry (r equals 0.44, p less than 0.0005) is as strong a predictor of disease progression as a positive cystoscopic examination (r equals 0.43, p less than 0.0005). The combination of bladder wash flow cytometry and voided urinary cytology is not superior to positive bladder wash flow cytometry alone. Median estimated interval to progression for these patients treated with bacillus Calmette-Guerin was 38 months. In the subgroup with positive bladder wash flow cytometry at 6 months the median interval to progression was 30 months. With a negative bladder wash flow cytometry at 6 months the probability of survival free of progression at 30 months was 85 per cent (p less than 0.01). Thus, negative bladder wash flow cytometry at 6 months is a strong predictor of response to bacillus Calmette-Guerin and also survival free of progression.     

Detection of carcinoma in the post-cystectomy urethral remnant by flow cytometric analysis

Following radical cystoprostatectomy for bladder cancer patients must be monitored for carcinoma of the urethral remnant. Cytological examination of urethral washings has proved to be a useful alternative to urethroscopy and urethrography. Urethral irrigation specimens also can be evaluated objectively by flow cytometry. In each of 4 patients in whom carcinoma developed in the urethral remnant analysis of urethral irrigation by flow cytometry was positive and comparable to results of urethral irrigation cytology. Flow cytometry is an objective and apparently reliable diagnostic test for malignancy in the urethral remnant.     

Monoclonal antibody 486 P 3/12: a valuable bladder carcinoma marker for immunocytology

Monoclonal antibodies directed against tumor-associated antigens of bladder carcinoma were used to identify tumor cells in bladder washout specimens of 40 patients with bladder carcinoma (group 1), 41 with no bladder disease or with urinary tract infections (group 2), 41 who received long-term mitomycin C instillation therapy after excision of the tumors (group 3) and 39 who received no prophylaxis after excision of the tumors (group 4). In all groups the same bladder washout specimen was used for standard urinary cytological and immunocytological tests. True positive results were obtained in 90 per cent of the patients in group 1 according to our immunocytological criteria and in 43 per cent according to standard cytology studies. No urine specimens in group 2 (controls) were immunocytologically positive, while 16 of 41 in group 3 and 17 of 39 in group 4 were positive immunocytologically but only 4 and 5, respectively, were positive according to standard cytology studies. Further followup of these patients will show whether cells positive for monoclonal antibody 486 P 3/12 will permit early detection of recurrent bladder cancer and whether one can identify patients who require prophylaxis after removal of the superficial bladder tumors.     

Primary squamous cell carcinoma of the male urethra: nuclear deoxyribonucleic acid ploidy studied by flow cytometry

Flow cytometry analysis was performed on 30 primary male urethral squamous cell carcinoma specimens. Nuclei were extracted from paraffin-embedded archival material and isolated nuclei were stained with propidium iodide. Bulbomembranous urethral tumors had a higher incidence of abnormal deoxyribonucleic acid ploidy patterns than penile urethral tumors (69 and 29 per cent, respectively). Of the tumors exhibiting a deoxyribonucleic acid diploid pattern and an abnormal (deoxyribonucleic acid tetraploid or aneuploid) histogram 18 and 93 per cent, respectively, showed tumor progression (p less than 0.001). None (0 per cent) of the low grade (grade 1 or 2) tumors with a deoxyribonucleic acid diploid pattern developed local recurrence or distant metastases, whereas 90 per cent of the low grade tumors with an abnormal deoxyribonucleic acid pattern progressed (p less than 0.002). Patients with tumors exhibiting deoxyribonucleic acid diploid ploidy had 5 and 10-year rates free of disease of 85 per cent. In contrast, patients with tumors with abnormal deoxyribonucleic acid ploidy patterns had 5 and 10-year rates of 20 and 0 per cent, respectively (p less than 0.001). Determination of deoxyribonucleic acid ploidy pattern by flow cytometry provides important prognostic information for male patients with primary squamous cell carcinoma of the urethra.     

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.     

Flow cytofluorometric DNA analysis in fresh bladder specimens and its correlations between cytology and mitotic index

From 1986 to 1987, cell materials of 50 patients with a suspicious history of bladder tumor were determined by flow cytofluorometric DNA measurement and compared with conventional cytogenetic analysis in the institution. The frequency of DNA aneuploid was found 47.6 per cent positive in 42 consecutive cases investigated by means of flow cytometry whereas in 47 cases by cytology and 42 cases by mitotic index (MI) profile a comparable 44.7% and 52.5% of positive findings were observed respectively, showing that there was good correlation between flow cytometry and conventional cytology. In a combined analysis of these 3 potential indices for tumor marker a 74 per cent positive rate could be reached and a good consistency of the 3 indices was observed by 69.7%. The study of DNA distribution pattern by FCM would definitely be of clinical significance in daily practice only if a disagreement appears a reevaluation should then be made. Cytological studies require a high level of skill and experience and it is time-consuming with subjective bias, comparing to FCM which sever as a valuable method with much more diagnostic sensitivity, objectivity and rapidity. Flow cytometry is now developed to the point as a particularly valuable adjuvant for bladder tumor detection.     

Occurrence of uroepithelial tumors of the upper urinary tract after the initial diagnosis of bladder cancer

We treated 519 patients with primary bladder cancer, of whom 12 had upper urothelial tumor during followup. Almost all patients had superficial bladder cancer at diagnosis. All but 1 of 12 patients who underwent total cystectomy with ileal conduit diversion also underwent various transurethral procedures for treatment of the primary bladder lesions. The over-all incidence of bladder cancer patients who subsequently had upper urinary tract tumors was 2.3 per cent. Among the patients with treated bladder tumors a higher incidence (13.2 per cent) was observed in dye workers than in the general population (1.1 per cent). The interval between initial treatment of the bladder cancer and diagnosis of the upper urinary tract tumor ranged from 7 to 170 months (mean 70 months). The frequency of upper urinary tract tumors increased with time. We conclude that the appearance of upper urinary tract tumor after diagnosis of primary bladder cancer may be promoted by nonspecific irritation of the urothelium, which previously was made unstable by urinary chemical carcinogens.     

Flow cytometry of the urinary bladder

Flow cytometry now appears sufficiently developed technically to identify carcinoma of the bladder on irrigation specimens with accuracy comparable to that of conventional exfoliative cytology. The cancer cells are distinguished by abnormal levels of DNA per cell, which can be quantified with acridine orange, a metachromatic fluorescent dye for DNA and RNA. With this technique it is also possible to follow the development of carcinoma and assess the effects of treatment by quantifying the number of cancer cells present in the irrigation specimen.     

Upper urinary tract tumors following bladder cancer

We treated in total 795 patients with primary transitional cell carcinoma of the urinary bladder between April, 1964 and December, 1988. Eighteen patients of them had upper urothelial cancer during the follow-up period. Thirteen of the 18 patients had received transurethral resection for the initial bladder cancer, while 3 total cystectomy and 2 segmental resection. The over-all incidence of bladder cancer patients who subsequently developed upper urinary tract tumors was 2.3 per cent. The interval between initial treatment of the bladder cancer and treatment for the upper urinary tract tumor ranged from 2 to 74 months (median 20 months). The five-year survival rate after treatment for the upper urinary tract tumor was 31.7 per cent. We conclude that the following are high risk patients for development of upper urinary tract recurrences: 1) patients with bladder cancer near orifices, 2) patients with recurrent bladder cancer under bladder preserving treatment for a long time, 3) patients with G2 multifocal bladder cancer.     

Chromosomal numerical aberrations detected by fluorescence in situ hybridization on bladder washings from patients with bladder cancer

OBJECTIVE: Previous studies on touch biopsy specimens have determined numerical or structural changes involving many different chromosomes in bladder cancer. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) assay in bladder washings as an objective technique to detect chromosomal numerical aberrations in bladder cancer. The main advantages of bladder washings are that they can be easily collected during the clinical follow-up of patients with superficial bladder cancer and they do not contain so many degenerate cells as urine samples. METHODS: We collected specimens from 25 patients who underwent transurethral resection of bladder tumors. Double target FISH assays with centromeric labeled probes for chromosomes 7, 8, 9 and 11 were used on the bladder washings and on the touch biopsy slides. The results were compared to flow cytometry and tumor grade and stage. RESULTS: We found monosomy 9 and trisomy 7, 8, 9 and 11 in 28, 32, 36, 28 and 25% respectively of the patients. FISH analysis of bladder washing versus touch biopsy specimens were concordant in approximately 90% of the slides. Total DNA aneuploidy correlated well with numerical aberrations of chromosomes 7, 8 and 11, but not with chromosome 9. CONCLUSION: Although better hybridization efficiency was obtained on touch biopsy slides, the results in bladder washings were in high concordance. FISH analysis on bladder washing samples may become a simple tool to improve the accuracy of cytology.     

Clinical application of NMP22 and urinary cytology in patients with hematuria or a history of urothelial carcinoma

For evaluation of the clinical application of immunoassay for nuclear matrix protein 22 (NMP22 immunoassay) and urinary cytology for early diagnosis and detection of bladder cancer in patients with hematuria and/or a previous history of bladder cancer, 209 urine samples obtained from 137 patients presenting episodes of hematuria or a history of bladder cancer were assayed for NMP22 levels and/or prepared for cytology examination. Biopsy was performed when any visible tumor was identified during cystoscopy examination. The median NMP22 concentrations measured in samples taken from patients with active bladder cancer, from patients with a history of bladder cancer but no active disease, from patients with hematuria, and from healthy volunteers were 18.95, 5.45, 6.39, and 3.75 U/ml, respectively. The urinary NMP22 level recorded for patients with urothelial carcinoma was significantly higher than that noted for individuals without active disease. The sensitivity of the NMP22 assay and of urinary cytology in diagnosing bladder cancer was 69% and 67%, respectively. In contrast, the specificity of these two diagnostic modalities reached 72% and 93%, respectively. The NMP22 assay is slightly more sensitive but less specific than urinary cytology in detecting bladder cancer. This study indicates that determination of urinary NMP22 levels is a useful and noninvasive tool for the detection of bladder cancer because of its high sensitivity. The urinary NMP22 assay may be used as a first-line routine screening method; however, it cannot replace the use of urinary cytology because of its lower specificity.     

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。