Another fluorescence-based cellular assay is the UroVysion assay (Abbott Molecular), which uses fluorescence in situ hybridization (FISH) to visually inspect chromosome copy numbers and specific DNA sequences directly in the cell nucleus.28,58
Genetic alterations resulting in bladder cancer are most frequently found in chromosomes 1, 3, 5, 7, 9, 11 and 17.28,59,60,61
The UroVysion assay uses a multitarget set of probes that hybridize to the centromeres of chromosomes 3, 7 and 17 and to the 9p21 locus of chromosome 9. The loss of the 9p21 locus, site of the p16 tumour suppressor gene, is the earliest and most frequent genetic aberration in bladder cancer.62
Recent published series on the FISH assay have demonstrated results in the range of 68.6%–100% and 65.0%–96.0% for sensitivity and specificity, respectively18,28,30,35,39,43,44,58,62,63
(). Thus the UroVysion assay, which has received FDA approval for the detection of recurrent bladder cancer, is more sensitive than urinary cytology but provides a similar, or slightly lower, specificity. In all studies, sensitivity increased with higher cancer grade. The FISH method examines changes at the nuclear level of the cell and is therefore unaffected by any benign conditions of the patient; it has been approved for use in patients with hematuria.30,35
Kipp and colleagues64
demonstrated that the UroVysion assay might be used to monitor the effects of BCG and other intravesicle therapies where a positive FISH assay results in a high likelihood for the progression to muscle-invasive disease.64
Variation in scoring criteria, the use of voided urine as opposed to bladder-wash urine, observer experience and sample stability and handling contribute to the variation of reported performance. Criticism of this method typically focuses on the high cost of the probes and the need for expensive equipment (fluorescent microscopes, specific filters, etc.) and trained technical personnel.28
The test is not point-of-care, and is time-consuming and not suited to high-throughput screening.
There is an important consideration with regard to false-positive results with the FISH test. Several studies have reported the detection of recurrent tumours within weeks or months after a false-positive test result. Although it is always possible that these tumours were originally overlooked during the original cystoscopy, others suggest that the assays are sensitive enough to detect changes occurring at the cellular level before the development of endoscopically visible lesions. Halling and colleagues18
have termed the latter phenomenon the “anticipatory positive” result; these authors reported recurrences in 7 of 13 patients within 3–12 months after a false-positive FISH score. Studies with other marker tests have also addressed the predictive value of false-positive results29,40,65
and include a study by Pode and colleagues,66
who discussed false-positive results with the BTA Stat test. However, Friedrich and colleagues67
have argued that false-positive BTA Stat tests can be expected, owing to the influence of many benign conditions, and that cystoscopy will often fail to detect small lesions and carcinoma in situ. Whereas other reports focused strictly on false-positive results, Friedrich and colleagues compared recurrences after false-positive and true-negative scores and determined that there was no significant difference among these groups with use of the BTA Stat, NMP22 Bladder Cancer Test or ImmunoCyt/uCyt+.67
Nevertheless, it remains advisable to closely monitor patients in the weeks and months after a false-positive test result.30
The current surveillance protocol for bladder cancer patients calls for cystoscopy with cytology every 3 months for the first 1–2 years, then every 6 months for 1–5 years and then yearly afterwards, provided no recurrent tumours are detected. This is a very costly endeavour and does not take into consideration the fact that not all patients are at equal risk for tumour recurrence.31
Although not powerful enough to replace cystoscopy, urinary biomarkers may be useful in extending the time between cystoscopic examinations because of their relatively high sensitivity and high NPV (especially for higher-grade lesions). In a recent study evaluating the ImmunoCyt/uCyt+ test, Lodde and colleagues31
ranked patients as low-, intermediate- or high-risk for tumour recurrence according to tumour size and initial stage and grade of the malignancy. Although 30 of 84 low-risk patients developed recurrent tumours within 3–96 months, all lesions were stage pTaG1, and there were no cases of progression into the muscle. These authors concluded that after resection and an initial cystoscopy at 3 months, a follow-up protocol of yearly cystoscopy and testing with ImmunoCyt/uCyt+ and cytology every 6 months could result in a significant cost savings without placing the patient at a greater risk for disease progression.31
Other recent studies have considered the economic impact of the use of urinary biomarkers, particularly in the screening of patients for bladder cancer. Such considerations are directly dependent on the incidence of the disease as well as on the sensitivity and specificity of the marker, in addition to the overall costs of each test.7
Lotan and colleagues6
reported that screening of all men aged 55 years or older was significantly more costly on a per-cancer basis than screening only in a high-risk group (more than US$400000 v. $3130). Svatek and colleagues7
suggest that marker screening of a high-risk group could be cost effective, although additional studies in an asymptomatic cohort are required before screening of a general population is recommended.
Although no ideal marker currently exists, a distinction should be made between the usefulness of a marker for the detection of de novo bladder tumours as compared with the monitoring of recurrent tumours in bladder cancer patients. For the detection of new tumours (e.g., in a screening population), a marker must have a high sensitivity for all tumours, even at the expense of a lower specificity. In the surveillance or monitoring setting, delayed diagnosis of a low-grade recurrent lesion will be unlikely to affect the patient's prognosis, and as such, the sensitivity for high-grade lesions would be more important. A test with a high sensitivity for high-grade tumours would result in a high NPV (for high-grade tumours), and this would be useful in prolonging the time interval between cystoscopies.