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Keywords:

  • UroVysion;
  • urine cytology;
  • bladder cancers;
  • urothelial cell carcinoma

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

BACKGROUND

Urine cytology has been used for screening of bladder cancer but has been limited by its low sensitivity. UroVysion is a multiprobe fluorescence in situ hybridization (FISH) assay that detects common chromosome abnormalities in bladder cancers. For this study, the authors evaluated the effectiveness of multiprobe FISH and urine cytology in detecting urothelial cell carcinoma (UCC) in the same urine sample.

METHODS

In total, 1835 cases with the following criteria were selected: valid results from both the multiprobe FISH assay and urine cytology in the same urine sample, histologic and/or cystoscopic follow-up within 4 months of the original tests, or at least 3 years of clinical follow-up information. The results of FISH and cytology were correlated with clinical outcomes derived from a combination of histologic, cystoscopic, and clinical follow-up information.

RESULTS

Of 1835 cases, 1045 cases were from patients undergoing surveillance of recurrent UCC, and 790 were for hematuria. The overall sensitivity, specificity, positive predictive value, and negative predictive value in detecting UCC were 61.9%, 89.7%, 53.9%, and 92.4%, respectively, for FISH and 29.1%, 96.9%, 64.4%, and 87.5%, respectively, for cytology. The performance of both FISH and cytology generally was better in the surveillance population and in samples with high-grade UCC. In 95 of 296 cases with atypical cytology that were proven to have UCC, 61 cases, mostly high-grade UCC, were positive using the multiprobe FISH assay.

CONCLUSIONS

The UroVysion multiprobe FISH assay was more sensitive than urine cytology in detecting UCC, but it produced more false-positive results. The current data suggest that the use of FISH as a reflex test after an equivocal cytologic diagnosis may play an effective role in detecting UCC. Cancer (Cancer Cytopathol) 2013;121:591–597. © 2013 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Bladder cancer is one of the most common cancers in the United States, with an estimated 73,000 new cases and greater than 14,000 deaths from bladder cancer in 2012.[1] Approximately 70% of bladder cancers are not muscle invasive, most of which are low-grade urothelial cell carcinomas (UCCs) that frequently will recur with a possibility of grade and stage progression.[2, 3] The reported recurrence rate of bladder cancers ranges from 15% to 61% at 1 year and from 31% to 78% at 5 year after initial treatment with fulguration or transurethral resection.[4, 5] Therefore, lifelong surveillance for recurrence of bladder cancer constitutes an important part of routine management in patients with bladder cancer. Urine cytology has been widely used in screening for bladder cancers. It is highly specific for the detection of high-grade UCC but suffers from low sensitivity, especially in cases of low-grade UCC.[6-8] The multiprobe fluorescence in situ hybridization (FISH) assay, UroVysion (Abbott Molecular Inc., Des Plaines, Ill), was developed to be more sensitive for detecting bladder cancers.[9] The assay was designed to detect the common chromosome abnormalities observed in UCC, ie, polysomy for chromosomes 3, 7, and/or 17 and homozygous loss of band 21 on the short arm of chromosome 9 (9p21) in urine samples. The UroVysion assay was approved by the US Food and Drug Administration initially for the surveillance of recurrent UCC and later was extended to detect bladder cancer in patients with hematuria.[9-11] Although the use of UroVysion as a supplement to urinary cytology has received generally favorable reviews, studies have demonstrated great variation in its sensitivity (range, 8%-100%) and specificity (range, 29%-100%) in detecting UCC.[12-18] Therefore, evidence-based studies regarding the effectiveness of the UroVysion multiprobe FISH assay and urine cytology in detecting UCC still are encouraged.[19] The objectives of the current study were to evaluate the sensitivity and specificity of UroVysion and cytology in same urine sample by correlating the histologic, cystoscopic, and clinical findings and to understand the clinical utility of the UroVysion assay.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

The study cases were identified by retrospective analysis of data from the Department of Pathology and Laboratory Medicine clinical laboratory database and the electronic medical record, with the following criteria: 1) valid results of both UroVysion and cytologic examination from same urine sample, including voided, catheterized, and bladder washing; and 2) histologic and/or cystoscopic follow-up within 4 months from the original tests; or 3) at least 3 years of clinical follow-up postlaboratory testing.

For cytologic studies, urine samples were processed with liquid-based technology (ThinPrep; Hologic, Inc., Bedford, Mass) and were read by 2 cytopathology board-certified pathologists who had a minimum of 10 years of experience in cytopathology. The original slides were not reviewed, and all cytologic diagnoses were obtained from the original cytology reports. For this study, cytologic diagnoses were classified into 3 categories: positive for UCC, atypical urothelial cells (AUCs), and negative for malignant cells. The cases with the original diagnosis of “suspicious for UCC” were included in the positive for UCC category, because the rates of malignancy in these 2 categories were similar in our institution based on the initial analysis of the data collected (70.8% in the positive category vs 69.6% in the suspicious category).

UroVysion tests were performed following the manufacturer's instructions, as previously described.[14] A positive test result was defined as 1 of the following: the presence of ≥4 morphologically abnormal cells of 25 analyzed cells that had polysomy of 2 or more chromosomes 3, 7, and 17 in the same cell; isolated gain of a single chromosome in ≥10% of cells; or homozygous deletion of 9p21 in ≥12 cells. The tests that produced unsatisfactory or equivocal results were excluded from the study.

To evaluate the performance of UroVysion and cytology in detecting UCC in the different population, the cases were divided into Hematuria group and Surveillance group; the former included the patients with hematuria or symptoms of urinary tract obstruction but no history of UCC, and the later included the patients with history of UCC. The performance of these 2 tests was also evaluated based on tumor grade. High-grade and low-grade UCC were determined based on the patients' history and/or follow-up histologic diagnoses. Patients with histological diagnosis of “carcinoma in situ” were included in high-grade UCC.

The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of UroVysion and cytology in detecting UCC were obtained by comparison to “gold standards,” defined as clinically positive and clinically negative for UCC, which were derived from the combination of the histological, cystoscopic, and clinical follow-up information. The criteria for clinically positive for UCC included: UCC confirmed by bladder biopsy performed concurrently or within 4 months of the tests; bladder lesions consistent with UCC in cystoscopic examination concurrently or within 4 months of the tests; and UCC diagnosed in outside institutions and referred to our institution for treatment. The criteria for clinically negative for UCC included: negative biopsy concurrently and/or within 6 months; or negative cystoscopic findings concurrently and no evidence of UCC within 1 year; or no evidence of UCC within 3 years by clinical follow-up which were obtained from patients' electronic medical charts.

The measures of sensitivity and specificity were compared between UroVysion and cytology using McNemar chi-square tests,[20] and the positive and negative predictive values (PPVs and NPVs) were compared between UroVysion and cytology using the Leisenring technique.[21] Both methods are ideal for measuring agreement between paired data. Statistical analyses were conducted using SAS v9.3 (SAS Institute, Inc., Cary, NC). The study was approved by the Institutional Review Board of the Medical University of South Carolina.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Between January 1, 2003 and December 31, 2006, 4782 urine samples were cytologically examined and all had valid cytologic diagnosis. Two thousand eight hundred seventy UroVysion tests were requested in the same period, and among them, 256 (8.9%) cases were not run because of low cellularity, and 38 (1.3%) cases received inclusive results. In total, 1835 cases from 957 patients (347 female and 610 male) met the search criteria and had valid results of both cytology and UroVysion from the same urine sample. The source of urine included 1112 void, 558 bladder washing, 98 catheterized, and 67 ileal conduit urine. Seven hundred ninety cases from 652 patients were tested for hematuria/symptoms of urinary tract obstruction, and 1045 cases from 305 patients were for surveillance of recurrent UCC. A total of 299 cases were determined to be Clinically Positive for UCC, and among them, 281 were confirmed by surgical biopsy performed concurrently and/or within 4 months of the tests; 10 had a bladder mass(es) seen in the concurrent cystoscopy; and 8 were diagnosed in outside institutions and referred to our institution for treatment. There were 1536 Clinically Negative cases: 245 had negative biopsy results within 6 months; 375 had negative concurrent cystoscopy and no evidence of UCC within 1 year; and 916 had no evidence of UCC within 3 years based on the information from electronic medical records.

The results from UroVysion tests and cytologic diagnoses in 1835 cases are listed in Table 1. Among 299 clinically positive cases, UroVysion was positive in 185 (61.9%) and cytology was positive in 87 (29.1%) cases; in 1536 clinically negative cases, UroVysion and cytology were negative in 1378 (89.7%) and 1287 (83.7%) cases, respectively. The false-positive and false-negative cases were 158 (10.3%) and 114 (38.1%), respectively for UroVysion, and 48 (3.1%) and 117 (39.1%), respectively for cytology. Two hundred ninety-six (16.1%) cases were cytologically diagnosed as AUC, 95 of them (32.1%) were clinically positive for UCC, including 52 cases of high-grade UCC and 43 cases of low-grade UCC. UroVysion was positive in 107 of 296 AUC cases: 61 (57.0%) cases were clinically positive for UCC, including 48 cases of high-grade UCC (Table 2). The sensitivity, specificity, PPV and NPV of UroVysion and cytology in detecting UCC were shown in Table 3. Comparing UroVysion to cytology, UroVysion had higher sensitivity (61.9% vs 29.1%; P<.0001) and NPV (92.4% vs 87.5%; P<.0001), and cytology had higher specificity (96.9% vs 89.7%; P<.0001) and PPV (64.4% vs 53.9%; P = .008). When stratified by the reasons for the tests, PPV of both UroVysion and cytology was higher in the surveillance group than in patients with hematuria, and NPV of both tests was higher in the hematuria group than in the surveillance group. For both tests, the sensitivity and specificity values were comparable between the 2 groups (Table 4). Both tests demonstrated higher sensitivity in detecting high-grade UCC when stratified by the tumor grade based on the patients' history and/or follow-up histological diagnosis (Table 5).

Table 1. Correlation of UroVysion and Cytologic Diagnoses With the Clinical Findingsa
Clinical FindingsNo. of CasesNo. of Cases (%)
UroVysion FISH DiagnosisUrine Cytology Diagnosis
PositiveNegativePositiveWith AUCsNegative
  1. Abbreviations: AUCs, atypical urothelial cells; FISH, fluorescence in situ hybridization.

  2. a

    UroVysion is a multiprobe FISH assay (Abbott Molecular Inc., Des Plaines, Ill).

Positive299185 (61.9)114 (38.1)87 (29.1)95 (31.7)117 (39.1)
Negative1536158 (10.3)1378 (89.7)48 (3.1)201(13.1)1287 (83.8)
Total183534314921352961404
Table 2. Correlation of a Cytologic Diagnosis of Atypical Urothelial Cells and UroVysion With the Clinical Findingsa
  UroVysion FISH: No. of Cases
Clinical FindingsNo. With AUCsPositiveNegative
  1. Abbreviations: AUCs, atypical urothelial cells; FISH, fluorescence in situ hybridization assay.

  2. a

    UroVysion is a multiprobe FISH assay (Abbott Molecular Inc., Des Plaines, Ill)

Positive956134
Negative20146155
Total296107189
Table 3. The Effectiveness of UroVysion and Urine Cytology for Bladder Cancer Detectiona
VariableSensitivity, %Specificity, %PPV, %NPV, %
  1. Abbreviations: AUCs, atypical urothelial cells; FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value.

  2. a

    UroVysion is a multiprobe FISH assay (Abbott Molecular Inc., Des Plaines, Ill).

  3. b

    P < .0001.

  4. c

    P < .01 compared with the same diagnostic test characteristic for UroVysion.

UroVysion FISH61.989.753.992.4
Urine cytology    
With AUCs: Negative29.1b96.9b64.4c87.5b
With AUCs: Positive60.983.8b42.2b91.7
Table 4. The Effectiveness of UroVysion and Urine Cytology for Detecting Bladder Cancer in Patients With Hematuria and a History of Urothelial Cell Carcinomaa
VariableSensitivity, %Specificity, %PPV, %NPV, %
  1. Abbreviations: AUCs, atypical urothelial cells; FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; UCC, urothelial cell carcinoma.

  2. a

    UroVysion is a multiprobe FISH assay (Abbott Molecular Inc., Des Plaines, Ill).

  3. b

    P < .01.

  4. c

    P < .0001.

  5. d

    P < .05 compared with the same diagnostic test characteristic for UroVysion.

  6. e

    P < .001.

Hematuria    
UroVysion FISH6093.435.597.5
Urine cytology    
With AUCs: Negative33.3b98.5c57.7d96.1b
With AUCs: Positive57.888.6e23.4b97.2
UCC    
UroVysion FISH62.286.259.287.7
Urine cytology    
With AUCs: Negative28.3c95.3c66.180.6c
With AUCs: Positive61.479.3c48.8e86.5
Table 5. The Effectiveness of UroVysion and Urine Cytology for Detecting Bladder Cancer in Patients With a History of Low-Grade and High-Grade Urothelial Cell Carcinomaa
VariableSensitivity, %Specificity, %PPV, %NPV, %
  1. Abbreviations: AUCs, atypical urothelial cells; FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; UCC, urothelial cell carcinoma.

  2. a

    UroVysion is a multiprobe FISH assay (Abbott Molecular Inc., Des Plaines, Ill).

  3. b

    P < .0001.

  4. c

    P < .001.

  5. d

    P < .01.

  6. e

    P < .05 compared with the same diagnostic test characteristic for UroVysion.

Low-grade UCC, n  =  468    
UroVysion FISH40.887.847.184.9
Urine cytology    
With AUCs: Negative14.3b96.8b53.881c
With AUCs: Positive45.980.2d38.1e84.9
High-grade UCC, n  =  576    
UroVysion FISH75.584.864.690.4
Urine cytology    
With AUCs: Negative36.8b94.1b69.580.2b
With AUCs: Positive71.278.3d54.7d88

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

Urine cytology is a noninvasive, inexpensive, and simple way to detect UCC. It is a valuable tool for detecting high-grade UCC, especially those flat lesions that are difficult to detect on cystoscopy.[22, 23] However, it suffers from generally low sensitivity with a range of 29% to 84%, which varies according to tumor grade and stage, with the lowest sensitivity seen in nonmuscle invasive low-grade tumors.[10, 24, 25] Accurate statistical analysis on urine cytology is very difficult owing to many factors, including very high recurrence rate and the existence of cases with cytologic diagnosis of AUC which, by definition, is an indeterminate and belongs to neither positive nor negative for UCC. Our data showed that the overall sensitivity and specificity of cytology in detecting UCC was 29.1% and 96.9%, respectively with AUC cases counted as negative in statistical analysis, and 60.9% and 83.8%, respectively with AUC counted as positive for UCC. While the statistical analyses with AUC as positive and negative are listed in the tables, for convenience, the discussion below will mainly be based on those with AUC counted as negative, consistent with most of previous studies.

In the current study, urine cytology had similar sensitivity and specificity for UCC detection in the patients with surveillance compared with those with hematuria and/or urinary obstruction symptom. The PPV for UCC detection was higher and the NPV was lower in the surveillance group compared with the hematuria group, as expected, mostly likely because of the difference in the prevalence of UCC between these 2 groups. When stratified by tumor grade, the sensitivity was 14.3% for low-grade UCC and 36.8 % for high-grade UCC.

The cytologic diagnosis of the low-grade UCC remains a challenge because of the lack of significant cytologic atypia and clear diagnostic criteria.[26, 27] Most urine samples with low-grade UCC show either no special cytologic features or only architectural abnormalities, ie, the presence of significant number of clusters of benign appearing and/or minimally atypical urothelial cells, which can also be seen in many other benign conditions, especially urinary tract lithiasis and catheterization. Other challenges in urine cytology include cellular atypia caused by reactive changes, such as those caused by human polyoma viral infection or prior therapy, including chemo-radiation and Bacillus Calmette-Guerin treatment related changes, which can mimic high-grade UCC. In addition, the experience and preference of a pathologist in urine cytology would certainly play a role in the outcome of urine cytology.[10] For these reasons, up to 1/3 of cases are placed in AUC category in routine practice.[27, 28] Our data indicated that the malignant rate was 70.4%, 32.1%, and 8.3% in cytologic diagnostic category of positive for UCC, AUC, and negative for UCC, respectively. The higher malignant rate in AUC category may partially explain the lower sensitivity of urine cytology in this study. Fortunately most low-grade UCC have a papillary growth pattern that can be easily detected by cystoscopic examination. Molecular tests, conversely, would be important ancillary tests for detection of high-grade UCC, especially the flat lesions that could be missed on cystoscopy.

UroVysion is a multiprobe FISH test performed on urinary cytology preparations, that identifies polysomies of chromosomes 3, 7, and 17 and locus-specific 9p21 deletions that are commonly seen in UCC. The test has been frequently studied and widely used in detection of UCC. Most of the studies comparing UroVysion and cytology concluded that UroVysion was superior to cytology in detection of UCC in almost all categories,[10-16] although a few showed the opposite results.[17, 29] In a meta-analysis of assay characteristics of UroVysion in comparison to cytology, Hajdinjak reported a pooled sensitivity and specificity of UroVysion were 72 and 83%, as compared with 42 and 96% for cytology.[30] UroVysion, like cytology, has been shown to have a lower sensitivity for the detection of low grade, as compared with high-grade UCC. False-positive UroVysion results have been attributed to the presence of abundant umbrella cells (could be uniform tetrapoloid cells) and human polyoma viral infection, while false-negative results could result from a low-grade UCC, paucity of cancer cells, therapy effects, obscuring inflammation, lubricant, and heavy squamous contamination of vaginal origin.[31-33] The data from our study showed the sensitivity and specificity of UroVysion was 75.6% and 84.8%, respectively for high-grade UCC, 40.8 % and 87.8%, respectively for low-grade UCC, and 61.9% and 89.7%, respectively for all UCC, which were similar to recently published studies.[11, 13] The variation in sensitivity and specificity of UroVysion in detecting UCC could be at least partially explained by the difference in study population, the sample size, and perhaps significant changes in demography of the patients. In earlier studies, the UroVysion assay was performed mainly on patients with a history of UCC with recurrent disease, including different proportions of invasive, high-grade, and low-grade UCC, therefore there was higher sensitivity and specificity. With extended utility to screen patients with hematuria and other symptoms, such as urinary tract obstruction, the sensitivity, specificity, PPV and NPV of the assay have reportedly been decreasing.[16, 26] Our data showed PPV was higher when UroVysion was applied in surveillance of recurrent UCC (59.2%) as compared with screening the patients with hematuria (35.5%), while NPV was higher in screening the patients with hematuria (97.5%), compared with surveillance (87.7%). However there was no obvious difference observed in the sensitivity and specificity of the assay between these 2 groups in this study. The sample size of the studies also had been a source of biases, as expected. The studies with the most extreme results were those that included only a small number of cases confounding the typical selection bias. Interestingly, a recent study demonstrated that the demography of patients tested had changed significantly in recent year with increased female patients with fewer true positive results, which could also have impact on the effectiveness of UroVysion test.[34] The current study represents 1 of the largest series on the utility of UroVysion, including 1835 cases with histological and/or detailed clinical follow-up information. Approximately 66% of patients (652 of 957) in the study were tested for hematuria and urinary tract symptoms, a population with lower prevalence of UCC, which might partially explain the lower sensitivity and PPV compared with the populations in previous studies.

Other potential applications of UroVysion include monitoring effectiveness of the treatments, predicting recurrence of UCC, and assisting with clarification of cytologically atypical cases via the use of a “reflex test.” Studies showed that patients with positive UroVysion test post BCG treatment was associated with failure of the treatment compared with those with negative result, and the patients with superficial bladder cancer who had positive UroVysion at the end of BCG treatment were at a higher risk for progression to muscle invasive disease.[35, 36] Thus, the patients with positive UroVysion test but negative cytology and cystoscopic findings were more likely to suffer from future recurrence and should be more closely monitored.[37, 38] Yoder et al observed that, over a period of 29 months, 65% of the cases with positive UroVysion but no visible lesions developed recurrent tumor on follow-up.[38] Our data indicated that 46% (158 of 343) of patients who had positive UroVysion tests did not develop UCC during up to the 3 year follow-up. Because currently no special management is available to these patients, the use of UroVysion in predicting recurrence may cause significant anxiety in both the patients and clinicians, limiting the clinical utility of the assay. In fact, the relatively high rate of false-positive and/or the ability of UroVysion to detect potential UCC years before the morphological changes could be detected have limited and may have partially contributed to the downward trend of clinician ordering UroVysion test in our institution. Use of UroVysion as a reflex test, ie, testing only on the cases with equivocal cytologic diagnoses, such as those with suspicious for malignant cells and AUC, has been studied.[16, 39-41] It has been reported that UroVysion analysis was useful in the clarification of equivocal or atypical cytology and a sensitivity of 100%, 89%, and 60% were obtained in patients with suspicious, atypical, and negative cytology, respectively, with the overall specificity was 97%.[16] A negative FISH test result under these situations likely correlated with benign cytological changes, although a low-grade UCC could not be excluded, because these tumors can also be negative with the UroVysion assay. In the current study, UroVysion test was positive in 48 of 52 high-grade UCC cases in AUC category, indicated that a clinical management algorism including UroVysion as a reflex test after equivocal cytology might be beneficial in detecting UCC for our patient population. It is noteworthy that, in this study, the results from UroVysion for UCC detection were similar to those from cytology when the AUC counted for positive, which could just be a coincidence.

In summary, we evaluated the characteristics of the UroVysion multiprobe FISH assay in detecting UCC compared with urine cytology in the same sample. In accordance with most previous studies, our data demonstrated that the UroVysion test had higher sensitivity and lower specificity compared with cytology, although the sensitivity of UroVysion was slightly lower than that in earlier studies. These results suggest that UroVysion should be reserved for patients with the highest risk, particularly those who have equivocal urine cytology results.

FUNDING SUPPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES

This project was supported by Award UL1TR000062 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. FUNDING SUPPORT
  8. CONFLICT OF INTEREST DISCLOSURES
  9. REFERENCES