J Alfred Witjes
Professor of Oncological Urology
Paula MJ Moonen
Department of Urology
Nijmegen Medical Centre
Bladder cancer is a disease with chromosomal abnormalities. Fluorescence in-situ hybridisation (FISH) detects these abnormalities in chromosomes 3, 7, 17 and the 9p21 locus. Data show the test as one of the most promising markers for bladder cancer. However, since large meta-analyses or reviews are absent, patient selection remains indefinite. FISH is still labour-intensive and costly, so the practical implementation remains low.
Nonmuscle-invasive bladder cancer
Nonmuscle-invasive bladder cancer covers a broad spectrum of tumours. At one end the relatively innocent pTaG1 tumours are seen as having a high risk of recurrence, while the risk of progression is small. At the other end the possibly life-threatening pT1G3 + CIS is found to be characterised by a high risk of recurrence and a high risk of progression. Consequently, frequent surveillance is necessary in all patients with nonmuscle-invasive bladder tumours.
Traditionally the surveillance regimen consists of combined cystoscopy (UCS) and cytology. Both methods have pros and cons. With cystoscopy even the low-grade papillary tumours are easily found. On the other hand, the procedure is invasive and a burden for the patient. Moreover, the high-grade carcinoma in-situ lesions can be completely imperceptible, even for highly experienced cystoscopists – as demonstrated by the use of intravesical 5-aminolevulinic acid (fluorescence cystoscopy).(1) Cytology has more of a chance to detect these flat lesions, although recent studies found a detection rate of only 60% of grade 3 lesions by cytology.
Also, does the accuracy of cytology appear to be influenced by the investigator? Is the result unreliable in cases of infection or after intravesical therapy? Is the sensitivity in low-grade lesions very low (11% for grade 1 and 31% for grade 2 lesions)?(2) Even though it is used as a gold standard in the follow-up of patients with nonmuscle invasive bladder cancer, this combination does not provide us with the tools for effective surveillance.
Fluorescence in-situ hybridisation
FISH has been known for several years in the diagnosis of bladder tumours. The test was approved by the US Food and Drugs Administration in 2001 for monitoring tumour recurrence in urothelial carcinoma. The cells that are examined are derived from urine or a bladder wash of the patient. The multitarget FISH analysis shows the enumeration of copies of (parts of) chromosomes in interphase (see Figure 1).
The test is composed of a combination of probes that yield the highest sensitivity and specificity for bladder cancer recurrence: the centromeric enumeration probes for chromosomes 3 (red), 7 (green), 17 (aqua) and the locus-specific identifier (LSI) probe for 9p21 (gold). Deletions within the short arm of chromosome 3 (3p) have been found in high-grade, muscle-invasive bladder cancer and numerical abnormalities of chromosome 7 are the most sensitive marker for urothelial cancer detection.(3) The p53 tumour suppressor gene was identified on chromosome 17, and a mutation in this TSG is associated with higher malignancy grade and stage. Homozygous deletion of the p16 gene at 9p21 is one of the most common alterations in urothelial cell carcinoma (UCC) and occurs early in the development of bladder cancer.(4) The FISH slide is scanned for morphologically abnormal cells – the same as considered in cytology. A minimum of 25 of these cells are viewed, and the test is considered positive if ≥4 of those cells exhibit gains of ≥2 chromosomes, or when in ≥12 cells a total loss of 9p21 signals is seen. So with FISH the molecular DNA changes are added to the conventional morphological changes.
Clinical analysis overview
For several years the FISH analysis has been studied. Most recently the results of nine studies performed between 2000 and 2004 evaluating the role of FISH in bladder cancer surveillance was reviewed by Jones.(5) Overall sensitivity of FISH was 74%. The sensitivity of FISH for grade 1, 2 and 3 tumours was 58%, 77% and 96% respectively. Similar findings occurred in stages, where the sensitivity for Ta, T1 and muscle-invasive carcinoma was 64%, 83% and 94% respectively. FISH outperformed conventional cytology across all stages and grades. Cytology detected only 67% of CIS, versus 100% detection by FISH. The specificity of FISH was comparable to that of cytology.
In 2005, van Rhijn et al reviewed four studies performed between 2000 and 2003 with patients under surveillance.(6) They found a median sensitivity of 79% (range 70–86) and a median specificity of 70% (range 66–93) for the FISH analysis. They considered the test as one of the most promising markers for surveillance at this time. The specificity of the FISH analysis appears to be comparable to that of cytology; in other words, it is not 100%. That means that there are patients with FISH positivity in the absence of concurrent detectable malignancy. However, a vast majority of these patients develop clinical evidence of tumours within several months. Therefore, the term “anticipatory positive” (AP) results has been introduced to replace the designation of “false-positive”. This means that FISH is positive in anticipation of the development of morphologic findings in a large majority of cases.
In addition to the use of the test in the surveillance of patients, several other applications have been considered. This is especially since the FISH result, unlike other urinary markers and conventional cytology, is not affected by haematuria, infection, instrumentation or intravesical therapy. One of the other applications is to use FISH to assess the response to intravesical therapy. Although this was studied in a small number of patients (37), Kipp et al found FISH useful in predicting recurrence and even progression based on a positive post-therapy FISH result.(7) Another approach is to use the test in the case of an atypical or negative cytology result. Skacel et al retrospectively tested 120 urine samples of patients with known malignancy.(8) They found that FISH detected 85% of the cancers that had been missed by conventional cytology. Bollmann et al classified the FISH results of their pilot study in three groups: negative, low risk and high risk, depending on the amount of chromosomal abnormalities in the sample.(9) Their results indicate that FISH may help to individualise the patient’s follow-up – identifying those patients at risk for progression while lowering the number of cystoscopies in patients with low-grade disease.
Although the presented results are very promising, clinical implementation remains low, and a clear-cut selection of the patient group that benefits most from this assay has not been determined. This can be caused by the absence of large meta-analyses or reviews. Small differences in the processing and/or scoring of the samples and especially differences in the selected patient groups make it difficult to compare different studies. Furthermore, a consensus for the criteria used for the evaluation of abnormal cells is lacking. And lastly, the test has a high workload and costs.
Although FISH currently appears to be one of the most sensitive and promising markers for bladder cancer, its use in the everyday practice of urologists is limited. The precise application and the patient group that benefits most from the test remains indefinite, and the clinical utility of the test remains low.
- Schmidbauer J, Witjes F, Schmeller N, et al. Improved detection of urothelial carcinoma in situ with hexaminolevulinate fluorescence cystoscopy. J Urol 2004;171:135-8.
- Halling KC, King W, Sokolova IA, et al. A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma. J Urol 2000;164:1768-75.
- Sokolova IA, Halling KC, Jenkins RB, et al. The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine. J Mol Diagn 2000;2:116-23.
- Williams SG, Stein JP. Molecular pathways in bladder cancer. Urol Res 2004;32:373-85.
- Jones JS. DNA-based molecular cytology for bladder cancer surveillance. Urology 2006;67 Suppl 1:35-45.
- van Rhijn BW, van der Poel HG, van der Kwast TH. Urine markers for bladder cancer surveillance: a systematic review. Eur Urol 2005;47:736-48.
- Kipp BR, Karnes RJ, Brankley SM, Harwood AR, Pankratz VS, Sebo TJ et al. Monitoring intravesical therapy for superficial bladder cancer using fluorescence in situ hybridization. J Urol 2005;173:401-4.
- Skacel M, Fahmy M, Brainard JA, 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 2003;169:2101-5.
- Bollmann M, Heller H, Bankfalvi A, et al. Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer? BJU Int 2005;95:1219-25.