Multiprobe FISH for enhanced detection of bladder cancer in voided urine specimens and bladder washings.

The aim of this study was to evaluate the UroVysion (Vysis, Downers Grove, IL) fluorescence in situ hybridization (FISH) test for improved detection of bladder cancer in urinary specimens. Three groups of specimens were examined, including voided urine specimens (1) collected before resection of bladder cancer, (2) from cystoscopically negative bladders of patients with previous bladder cancer, and (3) from patients with benign prostatic hyperplasia (controls). FISH positivity was defined as more than 2 urothelial cells with an abnormal signal copy number of at least 1 of the 4 probes. FISH was positive in 1 of 27 control specimens and in 33 (73%) of 45 pTa, 12 (100%) of 12 pT1, and 13 (100%) of 13 pT2-4 tumors. The results were similar in a series of 68 bladder washings. In addition, FISH of voided urine specimens was positive in 5 of 10 patients with negative follow-up cystoscopy results. Subsequent recurrence was found in 4 of these patients but in none of 5 patients with FISH-negative results. Multiprobe FISH markedly improves the sensitivity and specificity of cytology for the detection of bladder cancer in urine specimens.     

Evaluation of the NMP22 test and comparison with voided urine cytology in the detection of bladder cancer

The purpose of this study was to assess the clinical performance of the NMP22 test and to compare it with that of voided urine cytology for the detection of bladder cancer. The NMP22 test was evaluated in two groups of patients. The first group was comprised of patients with histologically confirmed active transitional cell carcinoma (TCC) of the bladder, and the second group contained those with a history of bladder TCC but that were considered to have no evidence of disease on the basis of cystoscopic evaluation of bladder and/or biopsy. Sensitivity was determined in voided urine samples from patients with active TCC of the bladder. Specificity was determined in the urine samples of patients with a history of bladder TCC but no current evidence of disease. The NMP22 test was positive in 53 of 70 samples from patients with active bladder TCC. The sensitivity of the NMP22 test (75.7%) is significantly better than that of voided urine cytology (55.7%). The specificity of the NMP22 test and of voided urine cytology were 72.2% and 88.9% respectively, in patients with a history of bladder TCC but no current evidence of disease. There was no significant difference between the specificity of NMP22 and that of urine cytology. The NMP22 test is superior to voided urine cytology in the detection of TCC of the bladder. The results of this study indicate that the NMP22 test is an useful adjunct to cystoscopy in the detection and monitoring of TCC of the bladder.     

多色荧光原位杂交检测尿沉渣诊断膀胱尿路上皮癌

目的研究应用多色荧光原位杂交检测尿沉渣诊断膀胱尿路上皮癌的可行性,并与尿脱落细胞学相比较。方法M-FISH法分析100例可疑膀胱尿路上皮癌或有相关病史患者和10例良性前列腺增生患者晨尿尿沉渣,同时行尿脱落细胞学检查,以病理为诊断标准。FISH探针是用随机引物法标记3、7、17号染色体着丝粒及9p21区带,统计染色体畸变组合诊断膀胱尿路上皮癌的阳性率。结果M-FISH低级别尿路上皮癌灵敏度75.6%,高级别尿路上皮癌为100%,总体灵敏度85.5%;尿脱落细胞学低级别尿路上皮癌灵敏度33.3%,高级别是96.0%,总体灵敏度62.9%,两者总体灵敏度之间有统计学差异(P<0.05);两者特异性分别是84.6%和87.8%,无统计学差异。结论M-FISH法检测灵敏度比尿脱落细胞学高,特异性相似,并可以诊断出所有的高级别浸润性肿瘤。     

Utility of the BTA stat test kit for bladder cancer screening.

Our study evaluated the BTA (bladder tumor antigen) stat test kit as a primary screening device for the detection of transitional-cell carcinoma (TCC) of the bladder, with direct comparison by voided urine cytology (VUC) on the same specimens. The unfixed voided urine of 100 patients with no history of bladder cancer who had signs and symptoms of dysuria, incontinence, and gross hematuria and microhematuria were tested using the one-step BTA stat test kit before processing via the cytospin technique for fluid cytological evaluation. The patients in the study were followed for up to 12 mo with repeated urine cytological testing, cystoscopy, and bladder biopsy when clinically indicated. Nineteen cases tested positive, and 81 cases tested negative on the BTA stat test. VUC diagnosed three cases as unequivocally positive for TCC, 93 cases as negative, and four cases in which unqualified atypical urothelial cells were noted. TCC was confirmed by cystoscopy and bladder biopsy in three of three cases diagnosed by VUC and in three of 19 cases that tested positive by the BTA stat test. These findings resulted in an 84% false-positive rate for the BTA stat test and no false-positive cases for VUC during the 12-mo follow-up period. The results indicate that the sensitivity and specificity of BTA stat test are comparable to those of VUC; however, owing to a relatively high false-positive rate, it can at best act as an adjunct to urine cytological study for bladder cancer screening.     

Urinary nuclear matrix protein 22 (NMP22): a diagnostic adjunct to urine cytologic examination for the detection of recurrent transitional-cell carcinoma of the bladder.

This study compares urine nuclear matrix protein 22 (NMP22) immunoassay and conventional urine cytologic examination for detecting recurrent transitional-cell carcinoma (TCC) of the urinary bladder. One hundred twenty-eight urine specimens from 107 patients with a history of TCC of the urinary bladder were studied. NMP22 immunoassay and conventional cytologic examination were performed on each specimen. The NMP22 and cytology results were then compared with the results of subsequent cystoscopies/surgical biopsies performed over a 6-mo follow-up period. The sensitivity of urine cytologic study for predicting recurrent TCC was 60%, while the sensitivity of NMP22 assay was 47%. When both NMP22 assay results and the cytologic interpretation were positive for TCC, the positive predictive value of the combined tests was 74%. When both tests showed negative results, the negative predictive power was 81%. Our findings suggest that urine NMP22 assay may represent a useful diagnostic adjunct to conventional urine cytologic examination for the detection of recurrent TCC of the urinary bladder.     

Urine: beyond cytology for detection of malignancy

In the present review we discuss various ancillary modalities for detection of malignancies in urine samples, with an emphasis on urothelial carcinomas. Flow cytometry, bladder tumor antigen (BTA), nuclear matrix protein (NMP), matrix metalloproteinase (MMP), human chorionic gonadotrophic (HCG), telomerase, and other techniques are discussed. DNA FCM is a relatively costly and sophisticated technique. It has a practical application in the diagnosis of bladder cancer among subjects at high risk and is of value in monitoring the course of the disease and anticipating recurrence following conservative treatment. The BTA test is a simple, rapid, and inexpensive adjunct to cystoscopy and the results of the test are equivalent or superior to those of voided urinary cytology. NMP-22 immunoassay is a useful diagnostic test for predicting recurrence of urothelial malignancy. It is also a cost-effective and sensitive screening test for detecting tumor in patients with urothelial carcinoma. Beta-HCG estimation in urine samples appears to be an efficient diagnostic marker for the assessment of distant metastasis in bladder carcinoma rather than a screening test. Other ancillary techniques such as detection of expression of cytokeratin 20 by RT-PCR, MMP-9 estimation, and fluorescent in situ hybridization and telomerase activity are rarely applied clinically in routine urinary samples and are not cost-effective.     

Non-radioactive measurement of telomerase activity in human bladder cancer,bladder washings,and in urine

Early diagnosis is still the most important prerequisite for successful cancer treatment and this holds true for bladder cancer. Urine cytology is commonly used as a non-invasive screening procedure for the detection of bladder carcinoma, but this method is labour-intensive and often generates false-negative results. The ribonucleoprotein telomerase appears to be a promising new cancer marker, since its activity has been reported to correlate with indefinite growth. The aim of this study was to investigate whether telomerase activity can be detected in bladder cancer and in corresponding bladder washings. For this purpose, a sensitive non-radioactive TRAP (telomeric repeat amplification protocol) detection system was developed. With this technique, telomerase activity was found in 95 per cent of the carcinomas (n=20), in 70 per cent of the corresponding bladder washings, but in none of the urine samples obtained from patients with bladder carcinoma. No telomerase activity was detectable in normal urothelium or in samples from dysplastic urothelium. The data obtained from bladder washings show that superficial carcinoma cells released into the bladder still harbour telomerase activity. The absence of telomerase activity in voided urine is thus most likely due to degradation or inactivation under these conditions. The high rate of telomerase activity in bladder carcinoma indicates that the activation of telomerase is a common step in the tumourigenesis of bladder cancer. © 1998 John Wiley & Sons, Ltd.     

Cytological significance of abnormal squamous cells in urinary cytology

The aim of this study was to evaluate the significance of abnormal squamous cells (ASCs) in urinary cytology to clarify whether finding of ASCs could improve diagnostic accuracy. A total of 3,812 urine specimens were reviewed. We focused on three parameters of ASCs, necrotic debris, and ASC clusters, and linked them to histological diagnosis and clinical information. ASCs were identified in 34 (0.9%) specimens from 21 different patients. The incidence of ASCs was higher in females than in males. The 34 urine specimens were categorized as voided urine (16 cases), bladder-catheterized urine (17 cases), and bladder-washed fluid (1 case). Six (28.6%) of 21 patients were histologically diagnosed as having combined urothelial carcinoma and squamous cell carcinoma (SCC). Eight patients (38.1%) were histologically diagnosed as having SCC originating from sites other than the urinary tract; those urine specimens showed ASCs that were likely to have been exfoliated from malignant lesions. Necrotic debris and ASC clusters were identified in 12 specimens (35.3%) from 11 patients and 4 specimens (11.8%) from 4 patients, respectively, from a total of 34 specimens. Our results indicate that a great amount of care is needed for cytological diagnosis when attempting to recognize ASCs in urine specimens because ASCs were identified in not only SCC of the bladder but also in carcinoma or nonmalignant lesions of nonurinary tracts. Necrotic debris was found not only in patients who had malignant bladder tumors but also in those who had malignant lesions in locations other than the bladder.     

Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine

Development of noninvasive methods for the diagnosis of transitional cell carcinoma (TCC) of the bladder remains a challenge. A ProteinChip technology (surface enhanced laser desorption/ionization time of flight mass spectrometry) has recently been developed to facilitate protein profiling of biological mixtures. This report describes an exploratory study of this technology as a TCC diagnostic tool. Ninety-four urine samples from patients with TCC, patients with other urogenital diseases, and healthy donors were analyzed. Multiple protein changes were reproducibly detected in the TCC group, including five potential novel TCC biomarkers and seven protein clusters (mass range, 3.3 to 133 kd). One of the TCC biomarkers (3.4 kd) was also detected in bladder cancer cells procured from bladder barbotage and was identified as defensin. The TCC detection rates provided by the individual markers ranged from 43 to 70% and specificities from 70 to 86%. Combination of the protein biomarkers and clusters, increased significantly the sensitivity for detecting TCC to 87% with a specificity of 66%. Interestingly, this combinatorial approach provided sensitivity of 78% for detecting low-grade TCC compared to only 33% of voided urine or bladder-washing cytology. Collectively these results support the potential of this proteomic approach for the development of a highly sensitive urinary TCC diagnostic test.     

Urine cytology of micropapillary carcinoma of the urinary bladder

A case of micropapillary carcinoma (MPC) of urinary bladder is presented, in which the urine smear was studied in detail in an attempt to better characterize the cytologic findings of MPC. When the voided urine was examined in low power, cancer cells were scattered in the specimens as compact papillary/spheroidal clusters composed of pleomorphic cancer cells. Solitary carcinoma cells were occasionally observed. High power view of the smear revealed that the papillae/spheroids consisted of high-grade urothelial carcinoma cells. The cancer cells had pleomorphic nuclei with coarsely granular chromatin and thickened, irregular nuclear membrane, and thick cytoplasm. Histologically, the tumor in the resected bladder appeared as small nests with surrounding hallo both in the luminal surface and in the site of wall involvement. These tightly bound papillary/spheroidal clusters comprised of highly atypical cancer cells were the most specific cytologic finding in the urine of MPC, which were considered as a key diagnostic clue of MPC. The background of the urine smear showed numerous granulocytes and bacilli compatible with cystitis, which is a previously known complication of MPC. Differential diagnoses of MPC from those with pertinent cytologic findings such as conventional UC (including glandular differentiation), and primary/secondary adenocarcinoma of urinary bladder are discussed with a brief review of literature.     

Tumor grading in urinary cytology. A study of the cytohistological relationship with evaluation of the kappa statistic for concordance

In order to verify both usefulness and reliability of cytologic grading evaluated on urinary sediment, we designed a study to test the ability to distinguish consistently between different degrees of neoplastic abnormalities of urothelial cells from voided urine, in relation to the histologic findings of bladder biopsies performed after cytologic reading. Kappa measures were calculated in 112 cytohistologic pairs representative of first disease detection (A) and in 35 pairs representative of disease recurrence (B). Cytology was able to correctly identify a cancer in 70.5% and 80.0% of group A and group B lesions, respectively. Unweighted kappa values of both groups A and B were poor (0.298 and 0.324 respectively). When we considered the weighted kappa, values suggesting fair agreement were obtained (0.576 and 0.500). Our data confirm that urinary cytology shows a good sensitivity in detecting bladder cancer, but it does not seem to give reliable results for the assessment of the tumor grading.     

Latent aggression-promoting properties of mouse bladder urine activated by heat

The aggression-promoting property of bladder urine in adult male mice was investigated. Under a newly developed stimulus application procedure (i.e., camel hairbrush applicator). Experiment 1 determined the minimal volume at which bladder and voided urine educed agonistic behavior in agressors. It was found that at least 0.05 ml of bladder urine and 0.03 ml of voided urine, both from intact males, were needed to induce significantly more attacks and uro-anal sniffs than respective urine types from castrated males. Also, bladder urine educed significantly less agonistic behavior than voided urine, both from intact males. Hence, the presence of a weak bladder urine chemosignal was confirmed. From these and previous findings, it was hypothesized that the chemocommunicative property of the weak bladder urine chemosignal could be accentuated by releasing the prochemosignal(s). Using 0.03 ml as the urine volume in Experiment 2, it was shown that heating bladder urine at 37 degrees C educed significantly more agonistic behavior than freezing bladder urine. The heating of bladder urine, however, effected significantly fewer attacks than the freezing of voided urine. It was concluded that bladder urine contains a latent and a free chemosignal, both of which are weak relative to voided urine chemosignal activity.     

Chromosomal alterations detected by fluorescence in situ hybridization in urothelial carcinoma and rarer histologic variants of bladder cancer

Fluorescence in situ hybridization (FISH) with the UroVysion probe set (Abbott Molecular, Des Plaines, IL) was used to assess 31 bladder cancers for chromosomal abnormalities, including 4 adenocarcinomas, 5 urachal adenocarcinomas, 6 small cell carcinomas, 7 squamous cell carcinomas, and 9 typical urothelial carcinomas. FISH was also used to assess the benign urothelium in 4 cases. There was a significant increase (P < .001) in the mean number of chromosome 3 (2.64 vs 1.51), chromosome 7 (2.61 vs 1.48), and chromosome 17 (2.41 vs 1.41) centromeric signals observed in cells from patients with cancer compared with patients without cancer. Of the 31 tumors, 29 (94%) demonstrated polysomic signal patterns in more than 10% of cells. In the 2 remaining tumor specimens, there was a high percentage of cells (>75%) demonstrating homozygous 9p21 deletion. The data from this study suggest that chromosomal abnormalities detectable by FISH in urothelial carcinoma are also common in rarer histologic variants of bladder cancer.     

In situ detection of global DNA hypomethylation in exfoliative urine cytology of patients with suspected bladder cancer

Global DNA hypomethylation is a common phenomenon in bladder cancer. Therefore we investigated whether it is possible to detect and assess global DNA hypomethylation in bladder cancer using a specific monoclonal antibody for 5-methyl-cytosine. Cytospins from exfoliative urine cytology specimens of patients with bladder cancer or a history of bladder cancer, control patients with benign urological diseases and of young healthy volunteers were analyzed. Urothelial carcinoma (UC) cells showed various degrees of nuclear destaining indicating global DNA hypomethylation whereas all specimens from healthy volunteers showed granular nuclear staining indicating regular methylation of repeated DNA sequences. Lowest 5-methylcytosine immunostaining scores were observed in carcinoma cells and a statistically significant difference was observed between urothelial cells of healthy controls or patients with benign disease compared to bladder cancer patients (p<0.01, p<0.05, respectively). In UC cases even morphologically normal urothelial cells often displayed evident hypomethylation. Likewise, in patients with a history of UC, but no cystoscopic evidence of recurrence, morphologically non-malignant urothelial cells presented with some degree of demethylation. Our results strongly support the hypothesis of early global demethylation in bladder cancer. Immunocytochemical staining with the 5-methylcytosine antibody allows simultaneous individual assessment of nuclear morphology and methylation status of a given sample.     

Real-time PCR assay for detection of quinolone-resistant Neisseria gonorrhoeae in urine samples

A need exists for the development of applicable surveillance tools to detect fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) in urine samples. We describe here a real-time PCR assay for detecting mutations in the Ser91 codon of the gyrA gene of N. gonorrhoeae in urine specimens. We tested 96 urine samples collected along with Gonorrhea Isolate Surveillance Project (GISP) urethral swab samples and compared the results with matched MICs of ciprofloxacin, as reported by the regional GISP laboratory. We then tested 100 urine specimens, known to be gonorrhea positive by nucleic acid amplification testing, provided by females to challenge the real-time PCR assay with urine specimens containing potentially less target DNA content than specimens from symptomatic males. With an MIC threshold of 0.125 mug of ciprofloxacin/ml, our assay correctly identified resistance in 41 of 44 (93.2%; 95% confidence interval [CI] = 81.3 to 98.6%) corresponding resistant culture specimens and correctly identified 51 of 51 (100%; 95% CI = 93.0 to 100%) susceptible specimens. One specimen did not amplify. The assay successfully amplified the gyrA amplicon and determined a susceptibility genotype in 72 of 100 (72%) urine specimens collected from female patients. We developed an assay for detecting QRNG in urine specimens that correlated well with MIC results of cultured specimens and had moderate sensitivity with urine specimens. This methodology might fulfill the need for a QRNG detection system for urine specimens, a useful characteristic in the age of nucleic acid amplification testing for gonococcal infection.     

A simple and practical technic for detecting cancer cells in urine and urinary bladder washings by flow cytometry

DNA measurement by flow cytometry has been demonstrated to be a potentially useful technic in the diagnosis of bladder cancer by detecting neoplastic cells in bladder washings and urine specimens. The authors' goal was to develop a simple and practical method utilizing the new generation of cytofluorographs designed for use in the clinical laboratory. This method combined direct fixation with cell lysis yielding fixed intact nuclei. Following RNase and pepsin digestion, the nuclei were separated from debris and aggregates on a sucrose barrier, stained with ethidium bromide, and analyzed with an argon laser analytic cytofluorograph. Urines and bladder washings from 14 patients with positive urinary cytology and histologically diagnosed bladder cancers were compared with specimens from patients without urothelial malignancies. DNA histograms clearly delineated aneuploid from diploid populations and often identified S, G2M, and G1 phase nuclei. Aneuploid populations have been detected in all tumor specimens with positive cytologies studied to date.     

Urine cytomorphology of micropapillary urothelial carcinoma

Micropapillary urothelial carcinoma (MPUC) is a rare subtype of urothelial carcinoma (UC) with an aggressive clinical course. The cytomorphologic features of MPUC in urine cytology have not been well described. In this study, 23 urine specimens (11 voided urines and 12 bladder washings) from 23 patients with MPUC on follow‐up surgical material and 28 specimens (14voided urines and 14 bladder washings) from 28 patients with high‐grade UCs (HGUC) were retrieved. Cytologic features (nuclear grade, cytoplasmic characteristics), architectural features (single cell pattern, true papillary structures, flat sheets/nests, three dimensional clusters, micropapillary (inside‐out, acinar‐like, or cauliflower with nuclei located peripherally)), and necrosis were evaluated. Clinical follow‐up was obtained by chart review. Two findings, micropapillae and cytoplasmic vacuoles, were seen more frequently in MPUC compared to HGUC, 81.0% vs. 14.3%, and 57.1% vs. 14.3%, respectively. The combination of these two findings had a sensitivity of 78%, a specificity of 86%, a positive predictive value of 82%, and a negative predictive value of 83% for the diagnosis of MPUC on subsequent biopsy. MPUC and HGUC can both exhibit a single cell pattern, papillary structures, flat sheets/nests, three dimensional clusters, high‐nuclear grade, and necrosis, thus these findings are not useful in distinguishing these entities. Chart review revealed that patients with MPUC had a higher rate of metastasis to lymph nodes and distant organs than HGUC, 57% vs. 4%. Therefore, the findings of cytoplasmic vacuoles and micropapillary structures in UC from a urine cytology specimen are associated with MPUC on subsequent biopsy. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

膀胱移行细胞癌中血型抗原Lewis A和Lewis X表达

目的　探讨血型抗原LewisA和LewisX在膀胱移行细胞癌中的表达及其诊断应用价值。方法　采用免疫组织化学EnVision方法 ,测定血型抗原LewisA和LewisX在 83例膀胱移行细胞癌和 6 8例非肿瘤性移行上皮黏膜中的表达 ,并收集10例膀胱癌及 10例正常人的尿脱落细胞标本进行LewisX免疫染色。结果　LewisA和LewisX在膀胱癌中的表达阳性率分别为 81 9% (6 8/ 83)和 83 1% (6 9/ 83) ,非肿瘤黏膜中的表达阳性率分别为 5 2 9% (36 / 6 8)和 11 8% (8/ 6 8)。LewisA和LewisX的表达强度与膀胱癌的病理分级和临床分期无关 (P >0 0 5 ) ,但膀胱癌的LewisA和LewisX表达强度高于非肿瘤黏膜 (P <0 0 5 )。 2 0例尿脱落细胞标本中 ,8例膀胱癌LewisX表达阳性 ,正常人组全部阴性。结论　LewisA和LewisX可成为诊断膀胱移行细胞癌的参考指标 ,特别是LewisX ,有助于低级别移行细胞癌的诊断     

Evidence of uncultivated bacteria in the adult female bladder

Clinical urine specimens are usually considered to be sterile when they do not yield uropathogens using standard clinical cultivation procedures. Our aim was to test if the adult female bladder might contain bacteria that are not identified by these routine procedures. An additional aim was to identify and recommend the appropriate urine collection method for the study of bacterial communities in the female bladder. Consenting participants who were free of known urinary tract infection provided urine samples by voided, transurethral, and/or suprapubic collection methods. The presence of bacteria in these samples was assessed by bacterial culture, light microscopy, and 16S rRNA gene sequencing. Bacteria that are not or cannot be routinely cultivated (hereinafter called uncultivated bacteria) were common in voided urine, urine collected by transurethral catheter (TUC), and urine collected by suprapubic aspirate (SPA), regardless of whether the subjects had urinary symptoms. Voided urine samples contained mixtures of urinary and genital tract bacteria. Communities identified in parallel urine samples collected by TUC and SPA were similar. Uncultivated bacteria are clearly present in the bladders of some women. It remains unclear if these bacteria are viable and/or if their presence is relevant to idiopathic urinary tract conditions.