Clinical Services

UroVysion® with Semi-Quantitative Analysis

Bladder cancer is the fifth most common cancer in the United States, with over 70,000 newly diagnosed cases and over 14,000 deaths annually.1 Bladder cancer is four times more likely to occur in men than in women. The median age at diagnosis is 65 years; bladder cancer is rare in individuals under 40.1 Ninety percent of bladder cancer cases are classified as transitional cell carcinomas (TCC), while the remaining 10% are predominantly squamous cell or adenocarcinomas.2 There are 4 clinically relevant subgroups of TCC, as defined by pathologic staging:

  • Carcinoma in situ (pTis)
  • Non invasive papillary TCC (pTa)
  • Minimally invasive TCC (pT1)
  • Muscle invasive tumors (pT2-pT4)

Each subgroup differs in clinical outcome.2-3 At presentation, 75% of tumors are non-muscle invasive (i.e., pTa, pT1 or pTis), of which 50 to 80% will have one or several recurrences, and 15 to 25% will progress to invasive tumors.4 For this reason, patients with non-muscle invasive bladder cancer are regularly monitored for tumor recurrence and progression with cystoscopy and sometimes urine cytology. Cystoscopy examination of the bladder, and often urine cytology, are also standard care for patients >40 years of age and presenting with hematuria. 5

Key issues in the clinical management of non-muscle invasive bladder cancer are the provision of adequate disease monitoring after resection of a primary tumor and prediction of the risk of recurrence and progression to invasive disease. Risk stratification of non-muscle invasive bladder cancer could facilitate more accurate characterization of divergent pathologic subtypes enabling individualized patient therapy and promoting the opportunity for tailored surveillance strategies.

Traditionally, indicators of an increased recurrence risk include high tumor stage and grade, size, multifocality, history of prior tumors and presence of carcinoma in situ (CIS). Intensity of surveillance is mostly individualized based on these clinical risk factors.6 The classic follow-up protocol after initial tumor resection consists of cystoscopic and cytologic examinations every 3 months for 2 years after tumor resection, then every 6 months for the following 2 to 3 years and then annually thereafter.7

Cystoscopy is an efficient method for the detection of primary or recurrent bladder cancer, but it is invasive and causes discomfort to the patient. Furthermore, flat tumors or CIS may be difficult to detect. Cytology has a high specificity in the detection of high-grade tumors that are not clearly evident on cystoscopic evaluation, including CIS and occult invasive cancers.8 However, sensitivity of cytology is not perfect in high-grade bladder cancer and even poor in low-grade urothelial neoplasia.9 Ancillary methods with enhanced sensitivity are therefore needed to better predict the individual risk of recurrence and progression, thereby helping clinicians determine which patients to treat more aggressively and which patients to treat with more conservative therapies and surveillance strategies.

Our understanding of the genetic changes that accompany bladder carcinoma initiation and progression are increasingly being elucidated.10-12 Homozygous deletions of the P16 gene at 9p21 locus are one of the most common alterations in bladder cancer and occur early in the development of both papillary tumors and carcinoma in situ.13-19 Bladder cancer progression is accompanied by increased chromosomal instability and aneuploidy.10,20-23 Cytogenetic studies reveal frequent alterations of a variety of chromosomes including chromosomes 1, 3, 7, 9, 17, and others.10

These chromosomal alterations can be detected with fluorescence in situ hybridization (FISH).24-30 FISH utilizes fluorescently labeled DNA probes to chromosomal centromeres or unique loci to detect cells with numerical or structural abnormalities indicative of malignancy. Studies have demonstrated that FISH, using the UroVysion® probe set, has similar specificity to and better sensitivity than routine cytology for detecting bladder cancer in urine.31-35 Presently, as approved by the US Food and Drug Administration (FDA), the UroVysion® assay is simply reported qualitatively as 'positive' or 'negative' for abnormality. However, recent studies indicate that assessing FISH quantitatively (i.e., pattern of chromosomal abnormality and percentage of abnormal cells) may help better predict which patients will recur and progress to muscle invasive disease.36-37

Given this quantitative information, patients with an anticipated uneventful clinical course and/or recurrent low-grade papillary tumors can be distinguished from those who are at a high risk of tumor progression. Accordingly, surveillance strategies could be tailored for individual patients on the basis of the biological properties of their disease.

Introduction to UroVysion®

The UroVysion® Assay is a FISH assay that was developed for the detection of cytogenetic evidence of urothelial neoplasia in urine specimens as an aid for initial diagnosis of bladder carcinoma in patients with hematuria and subsequent monitoring for tumor recurrence in patients previously diagnosed with bladder cancer. UroVysion® received FDA approval for the detection of recurrent tumor in patients with a history of bladder cancer in 2001 and for the detection of bladder cancer in patients with microhematuria but no previous history of bladder cancer in 2005.

It consists of fluorescently labeled DNA probes to the pericentromeric regions of chromosomes 3 (SpectrumRed), 7 (SpectrumGreen), 17 (SpectrumAqua) and to the 9p21 (SpectrumGold) locus containing the P16 tumor suppressor gene. When hybridized and visualized, these probes provide qualitative and semi-quantitative information on DNA alterations relating to the detection of active and/or potential urothelial neoplasia and the differentiation of low-risk and high-risk non-muscle invasive bladder cancer. With the assay result and taking into account other standard diagnostic procedures, the physician and patient can make more informed decisions regarding the initial diagnosis and subsequent surveillance of bladder cancer.

Criteria for UroVysion® Abnormality

The normal complement of FISH signals for non-neoplastic urothelial cells is two signals for each of the 3 centromeric (CEP 3, CEP 7, and CEP 17) and locus-specific (LSI 9p21) probes. However, a small fraction (< 10%) of normal cells may show only one copy of one or more of the four probes due to signal overlap or incomplete hybridization. Urothelial carcinoma cells, on the other hand, will show various patterns of aneuploidy for chromosomes 3, 7, 17 and loss of the 9p21 locus.

  • Gains of multiple chromosomes: cells that show gains (>= 3 copies) for two or more of the three centromeric (CEP 3, CEP 7, and CEP 17) probes.
    • Non-tetrasomy: cells that show gains (>=3 copies) for two or more of the three centromeric (CEP 3, CEP 7, and CEP 17) probes but not meeting the requirement for tetrasomy/near-tetrasomy (e.g., # of signals CEP3, CEP7, CEP17, LSI 9p21: 3-5-4-2; 4-4-5-1; etc.)
    • Tetrasomy: cells that show four copies for each centromeric (CEP 3, CEP 7, and CEP 17) probes (e.g., # of signals CEP3, CEP7, CEP17, LSI 9p21: 4-4-4-4).
    • Near-tetrasomy: cells that show four copies for any two of the centromeric (CEP 3, CEP 7, and CEP 17) probes but three copies for one (e.g., # of signals CEP3, CEP7, CEP17, LSI 9p21: 3-4-4-4; 4-3-4-4; 4-4-3-4)
  • Gains of a single chromosome: cells that show an isolated gain for one of the three centromeric (CEP 3, CEP 7, and CEP 17) probes.
    • Trisomy: cells that show three copies for one of the three centromeric (CEP 3, CEP 7, and CEP 17) probes but two or fewer copies of the other two probes (e.g., # of signals CEP3, CEP7, CEP17, LSI 9p21: 2-3-2-2; 3-2-2-1; etc.).
    • Non-trisomy: cells that show gains (>=4 copies) for one of the three centromeric (CEP 3, CEP 7 or CEP 17) probes (e.g., # of signals CEP3, CEP7, CEP17, LSI 9p21: 4-2-2-2; 2-5-2-1; etc.).
  • 9p21 loss: cells can show a homozygous or heterozygous loss of 9p21 signals.
    • Homozygous loss (9p21): absence of both 9p21 signals (e.g., # of signals CEP3, CEP7, CEP17, LSI 9p21: 2-2-2-0; 2-1-2-0; etc.).
    • Heterozygous loss (9p21): presence of only one 9p21 signal (e.g., # of signals CEP3, CEP7, CEP17, LSI 9p21: 2-2-2-1; 2-1-2-1; etc.).

A specimen is considered 'positive' for FISH-associated evidence of urothelial carcinoma if at least one of the following criteria is met:

  1. >=4 non-tetrasomic cells showing gains for 2 or more chromosomes 3, 7, and 17 in the same cell;38 or
  2. >=10 cells showing tetrasomy/near-tetrasomy for chromosomes 3, 7 and 17;38 or
  3. >=10 cells showing gains for a single chromosome 3, 7 or 17;39 or
  4. >=12 cells with homozygous loss of the 9p21 locus.

In addition, FISH results can be classified into 'low-risk' and 'high-risk' groups for prognostic analysis. Based on the chromosomal pattern and percentage of abnormal cells, low-risk patients with an anticipated uneventful clinical course and/or recurrent low-grade papillary tumors can be distinguished from those high-risk patients who are more prone to tumor progression.

  1. Low-risk FISH:
    1. Negative-FISH result; or
    2. Positive-FISH result:
      1. >=5% cells demonstrating non-tetrasomic gains for 2 or more chromosomes 3, 7, and 17 in the same cell;37 or
      2. >10% cells showing tetrasomy/near-tetrasomy for chromosome 3, 7, and 17;39 or
      3. >=10% cells showing gains for a single chromosome 3, 7 or 17;39 or
      4. 9p21 loss39
  2. High-risk FISH:
    1. Positive-FISH result:
      1. >=5% cells demonstrating non-tetrasomic gains for 2 or more chromosomes 3, 7, and 17 in the same cell (bladder cancer recurrence);37 or
      2. >10% cells demonstrating non-tetrasomic gains for 2 or more chromosomes 3, 7, and 17 in the same cell (progression to muscle-invasive cancer)37
      3. >=10% cells showing tetrasomy/near-tetrasomy for chromosome 3, 7, and 17 (bladder cancer recurrence and progression);39 or
      4. >=10% cells showing gains for a single chromosome 3, 7 or 17 (bladder cancer recurrence and progression)39

UroVysion® Images

The following examples of patient specimens demonstrate both the clinical utility of the UroVysion® Assay as well as its simplicity of interpretation.

UroVision Figure 1 Figure 1: Normal result observed in an urothelial cell after hybridization with the UroVysion® FISH probe showing two chromosome 3 (red), two chromosome 7 (green), two chromosome 17(aqua) and two P16 (gold) signals.
UroVision Figure 2 Figure 2: Abnormal result observed in an urothelial cell after hybridization with the UroVysion® FISH probe showing four chromosome 3 (red), three chromosome 7 (green), four chromosome 17(aqua) and two P16 (gold) signals.

Comparison of Cytology and UroVysion® for the Detection of Bladder Cancer

UroVysion® has a significantly higher sensitivity than cytology (81% vs. 58%) for the detection of bladder cancer, and clarifies cases in which the cystoscopy or cytology results are equivocal or suspicious (Table 1).41 The specificity of UroVysion® is approximately 96% among healthy and non-healthy subjects, which translates to fewer false positives.

Figure 4

Cystoscopy in Combination with UroVysion®

Based on the finding that FISH is more sensitive than conventional cytology and cystoscopy, there has been some interest in FISH replacing both. However, none of these modalities alone approaches the synergy of cell-based testing combined with endoscopy.

When reviewing the combination of cystoscopy with either cytology or UroVysion®, Halling et al.41 found that the combination of cystoscopy and conventional cytology detected 88% of tumors identified in their series at the Mayo Clinic, whereas the combination of cystoscopy and molecular cytology (conventional cytology combined with UroVysion®) detected 97%. Thus, the traditional combination was better than either test alone, but it missed >=4 times more cases of bladder cancer than did the latter combination using molecular cytology.

Thus, it is feasible that molecular cytology may replace conventional cytology in many practices, but it will be unlikely to ever offer an alternative to cystoscopy. It is more likely that FISH testing will allow the urologist to differentiate high-risk from low-risk patients at the time of their initial surveillance cystoscopy and therefore may allow tailoring of the surveillance protocol (including cystoscopy) to that risk.

Quantitative Molecular Cytology in the Surveillance of Non-muscle Invasive Bladder Cancer

Numerous studies have shown that FISH, using the UroVysion® probe set, has comparable specificity to and better sensitivity than routine cytology in the detection of bladder cancer in urine specimens.31-35 The UroVysion® assay, as currently performed by most reference laboratories, is simply reported qualitatively as positive or negative for abnormality. However, mounting data suggests that assessing FISH quantitatively may help to stratify the risk of recurrence and progression of non-muscle invasive bladder cancer.31,42-43

A recent study by Kruger et al. showed numerical changes of chromosome 17 and the 9p21 locus to be independent predictors of stage Ta tumor recurrence. A separate prospective study of intermediate-risk patients (those with T1G2, TaG2, or multifocal TaG1 lesions) found that 60% of those with aneuploidy of chromosome 7 and/or 17 developed recurrence. However, only 15% of patients with numerical alterations of chromosome 3 and/or the 9p21 locus had recurrence, indicating that some alterations may be more significant than others.43

In a retrospective cohort study, Kipp et al. demonstrated that among patients with a history of non-muscle invasive bladder cancer there is a positive correlation between the percentage of polysomic cells found in the urine by FISH and bladder cancer recurrence and progression to muscle invasive disease.37 The percent abnormal by FISH was one of the most significant variables (HR 1.026, P < 0.001) for predicting recurrent bladder cancer with a 2.6% increased chance of having cancer for every 1% increase in the percentage of abnormal cells by FISH. Similarly, the percentage of abnormal cells was one of the most significant (P < 0.001) variables for identifying disease with a 1.8% increased risk of having muscle invasive cancer for every 1% increase in the percentage of abnormal cells by FISH.

Anticipatory Positive Results

The FISH assay is quite sensitive, and it is not uncommon for the assay to be positive for a patient in whom tumor cannot be identified. Several studies have now demonstrated that FISH can detect recurrent urothelial carcinoma before it is clinically evident by cystoscopy.31-32,34,41-42 Sarosody et al. reported that there were 36 patients with a negative cystoscopic examination but a positive FISH result.31 With continued longitudinal follow-up, 15 (42%) of these cases were found to have biopsy-proven tumor recurrence, with time to tumor diagnosis of 3 to 16 months (mean, 6.0 months). Conversely, among 68 patients who had a negative cystoscopy and a negative FISH result, only 13 (19%) had a biopsy-proven recurrence at 3 to 19 months (mean, 11.2 months). The patients with a positive FISH result but negative cystoscopy were referred to as "anticipatory positive" cases. A Kaplan-Meier curve showed that the time to tumor recurrence was significantly less for patients with anticipatory positive FISH results compared with those with negative FISH results. A recent report by Yoder et al.32 found that approximately 27% of patients with a negative or atypical cytology result had a positive FISH result but no evidence of tumor. However, approximately 65% of these patients were found to have tumor recurrence within 29 months, which further suggests that positive FISH results cannot be ignored despite an absence of clinically detectable tumor.

Monitoring Intravesical Therapy

Surveillance cystoscopy and cytology after Bacillus-Calmette Guerin (BCG) therapy can be difficult to interpret due to inflammatory changes in the urothelium. Kipp et al. used FISH to assess the response to therapy in patients with superficial bladder cancer receiving BCG or other intravesical therapies.36 This study demonstrated that BCG did not interfere with the interpretation of FISH results and that FISH was able to identify patients that had a higher risk of tumor recurrence and muscle invasive disease. Patients with a positive FISH result at the end of their treatment were 4.6 times more likely to develop recurrent tumor and 9.4 times more likely to develop muscle-invasive tumor than patients with a negative result.

Risk Stratification and Surveillance Strategies

Ongoing experience with the UroVysion® FISH assay suggest that the pattern of chromosomal aneuploidy and percent abnormal cells in a positive FISH assay itself may have clinical significance.37,40 The results obtained with the FISH may be used not only to detect FISH-associated evidence of urothelial cancer but also to distinguish between patients with low-risk and high-risk non-muscle invasive bladder cancer. Based on the chromosomal pattern and percentage of abnormal cells, low-risk patients with an anticipated uneventful clinical course and/or recurrent low-grade papillary tumors can be distinguished from those high-risk patients who are more prone to tumor progression. More aggressive assessments and a shortened interval between examinations may be advisable for patients with high-risk FISH results, whereas patients with low-risk FISH results could undergo longer intervals between invasive cystoscopic monitoring.

The UroVysion® with Semi-Quantitative Analysis Advantage

  • Detects chromosomal abnormalities associated with the development and progression of bladder cancer.
  • UroVysion® in conjunction with cystoscopy delivers the best balance of sensitivity and specificity.41
  • UroVysion® is not affected by BCG immunotherapy and may predict response to intravesical therapy.36
  • Detects bladder cancer recurrence up to 6 months sooner than current diagnostic methods.
  • Aid to better distinguish between patients with low-risk and high-risk non-muscle invasive bladder cancer.

Ordering

Please contact BioVantra Client Support Center at (866) 301-0960 to arrange for UroVysion® testing. Our experienced Client Support Team can assist with:

  • Specialized Testing Requisition
  • Sample requirements
  • Logistics and specimen transportation
  • Testing methodology
  • Report delivery, status or interpretation
  • Expert oncology and pathology consultations
  • Requests for BioVantra literature and scientific references

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References

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  31. Sarosdy MF, Schellhammer P, Bokinsky G, Kahn P, Chao R, Yore L, Zadra J, Burzon D, Osher G, Bridge JA et al.: Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer. J Urol. 168: 1950-4, 2002.
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  33. Varella-Garcia M, Akduman B, Sunpaweravong P, Di Maria MV and Crawford ED: The UroVysion fluorescence in situ hybridization assay is an effective tool for monitoring recurrence of bladder cancer. Urol Oncol. 22: 16-9, 2004.
  34. Skacel M, Fahmy M, Brainard JA, Pettay JD, Biscotti CV, Liou LS, Procop GW, Jones JS, Ulchaker J, Zippe CD et al.: Multitarget fluorescence in situ hybridization assay detects transitional cell carcinoma in the majority of patients with bladder cancer and atypical or negative urine cytology. J Urol. 169: 2101-5, 2003.
  35. Skacel M, Pettay JD, Tsiftsakis EK, Procop GW, Biscotti CV and Tubbs RR: Validation of a multicolor interphase fluorescence in situ hybridization assay for detection of transitional cell carcinoma on fresh and archival thin-layer, liquid-based cytology slides. Anal Quant Cytol Histol. 23: 381-7, 2001.
  36. Kipp BR, Karnes RJ, Brankley SM, Harwood AR, Pankratz VS, Sebo TJ, Blute MM, Lieber MM, Zincke H and Halling KC: Monitoring intravesical therapy for superficial bladder cancer using fluorescence in situ hybridization. J Urol. 173: 401-4, 2005.
  37. Kipp BR, Tanasescu M, Else TA, Bryant SC, Karnes RJ, Sebo TJ and Halling KC: Quantitative fluorescence in situ hybridization and its ability to predict bladder cancer recurrence and progression to muscle-invasive bladder cancer. J Mol Diagn. 11: 148-54, 2009.
  38. Zellweger T, Benz G, Cathomas G, Mihatsch MJ, Sulser T, Gasser TC and Bubendorf L: Multi-target fluorescence in situ hybridization in bladder washings for prediction of recurrent bladder cancer. Int J Cancer. 119: 1660-5, 2006.
  39. Bollmann M, Heller H, Bankfalvi A, Griefingholt H and Bollmann R: Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer? BJU Int. 95: 1219-25, 2005.
  40. Halling KC, King W, Sokolova IA, Meyer RG, Burkhardt HM, Halling AC, Cheville JC, Sebo TJ, Ramakumar S, Stewart CS et al.: A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma. J Urol. 164: 1768-75, 2000.
  41. Bubendorf L, Grilli B, Sauter G, Mihatsch MJ, Gasser TC and Dalquen P: Multiprobe FISH for enhanced detection of bladder cancer in voided urine specimens and bladder washings. Am J Clin Pathol. 116: 79-86, 2001.
  42. Pycha A, Lodde M, Comploj E, Negri G, Egarter-Vigl E, Vittadello F, Lusuardi L, Palermo S and Mian C: Intermediate-risk urothelial carcinoma: an unresolved problem? Urology. 63: 472-5, 2004.
  43. Kruger S, Mess F, Bohle A and Feller AC: Numerical aberrations of chromosome 17 and the 9p21 locus are independent predictors of tumor recurrence in non-invasive transitional cell carcinoma of the urinary bladder. Int J Oncol. 23: 41-8, 2003.