Fluorescence in situ hybridisation (FISH) can identify urothelial carcinoma cells naturally exfoliated into the urine and its clinical use constitutes both a surveillance tool and an accurate diagnostic assay
Lorenza Pecciarini PhD
Molecular Oncologic Diagnostic
Laboratory – Pathology Unit
San Raffaele Scientific Institute, Milan Italy
Email: [email protected]
Most solid tumours, including urothelial carcinoma (UC), are characterised by numerical and structural chromosomal abnormalities. While normal cells are typically diploid (two copies of each gene), malignant cells can exhibit extra copies or deletions of large chromosomal regions and/or single genes. Chromosomal gains involving chromosome 3, 7, and 17 are commonly present in malignant urothelial cells, while 9p21 harbours p16, a tumour suppressor gene, that is often lost in these abnormal cells.1,2
Because urothelial carcinoma cells readily exfoliate into the urine, molecular cytogenetic techniques have been used to detect cells that have the chromosomal abnormalities consistent with a diagnosis of UC and this approach has proven useful for the identification of atypical cells well before morphological changes are apparent through cytological and histological microscopic examination.1
In particular, fluorescence in situ hybridisation (FISH) is one of the molecular cytogenetic standard methods for detecting genetic alterations in interphase nuclei by using fluorescently labelled DNA probes. Two types of FISH probes can be used for this purpose: chromosome enumeration probes, which hybridise pericentromeric regions of chromosomes, and locus-specific indicator probes, which hybridise to single genes and specific chromosomal regions of interest. The number of copies of a given probe target in the nucleus of a cell are then estimated under fluorescence microscopy.3
The UroVysion® test
A multi-target FISH test named UroVysion® Bladder Cancer Kit (Abbott Molecular, Inc., Des Plaines, IL), was first approved by the US Food and Drug Administration (FDA) in 2001 to monitor bladder cancer recurrence by analysing genetic alterations of the urothelial cells found in void urine; in 2005 the assay was expanded as a screening test for patients with haematuria and no cancer history.
It consists of four multicolour probes, 3, 7 and 17 centromeres probes (labelled with SpectrumRed, SpectrumGreen and SpectrumAqua, respectively) and p16 (9p21) locus (labelled with SpectrumGold) that detect the above-mentioned most common chromosomal aberrations associated with UC.4–6
Under a fluorescence microscope with appropriate objectives and filters, the observer needs to count the 4-colour fluorescent signals that assess the copy number of each target in the cell nuclei.
To date, there are no uniform criteria for urine FISH scoring7 but the test is generally considered positive following the UroVysion criteria (see UroVysion package insert): 25 urothelial cells must be selected based on nuclear abnormalities including enlargement, irregular borders and patchy nuclear staining with the nuclear counterstain 4-6-diamidino-2-phenylindole (DAPI) and the signal distribution for morphologically abnormal cells showing either a gain of multiple chromosomes (three or more signals) for more than one of centromere 3, centromere 7, or centromere 17 probes or a homozygous loss of p16 (no signals for locus 9p21) is recorded.
Analysis continues until either ≥4 cells with gains of multiple chromosomes or ≥12 cells with homozygous loss of 9p21 are detected, or until the entire sample is analysed. The total numbers of chromosomally abnormal cells (cells with gains of multiple chromosomes or homozygous loss of 9p21) are determined and the final results are reported as positive or negative (see Figure 1).
Fig. 1: FISH performed on voided urine samples hybridised with ZytoLight SPEC p16/CEN 3/7/17 Quadruple Color Probe, Zytovision: (A) normal cell showing two chromosome 3 centromere signals (red), two chromosome 7 centromere signals (green), two chromosome 17 centromere signals (aqua) and two 9p21 locus signals (yellow); (B) abnormal cells showing polysomy (more than four copies) of chromosome 3, 7 and 17 centromeres; (C) abnormal cells showing homozygous deletions (loss of the two normal copies) of the 9p21 locus (yellow).
Uncertain urine FISH results
Studies carried out on urine specimens from normal patients reveal that about 5–10% of cells show only one copy (monosomy) of a given probe (both centromeres and locus-specific one), and a small percentage of cells (1–3%) can show three (trisomy) or four (tetrasomy) copies of one or more centromere probes.
The finding of normal cells with monosomy is attributed to the fact that two signals may overlap and appear as one signal or that hybridisation may not be 100% efficient, while occasional cells with trisomy or tetrasomy may be in the DNA replication S or G2 phases of the cell cycle. Therefore it has been suggested that the presence of small numbers of cells with monosomy, trisomy, or tetrasomy may not be evidence of neoplasia. However, an abundance of cells with trisomy, tetrasomy, and even monosomy may be indicative of neoplasia.5
In our practice, over 90% of the cases that are called positive demonstrate polysomic (more than four copies of the centromeres probes) signal patterns, which generally correlate with presence of a high grade tumour. To our knowledge at the moment little is reported about the clinical correlations of cases showing only trisomy or tetrasomy and more data is still needed about the real meaning of these kind of findings.
Urine FISH sensitivity
Several studies described how the urine FISH test has increased sensitivity over cytology alone: this assay was proven to have a sensitivity of 90–100% for the detection of invasive bladder cancer (pT1–4) and a specificity of 95%; in the low-grade non-invasive bladder cancer, FISH increased the sensitivity of cytology from 25% to 60–75%. A meta-analysis of published studies reported the sensitivity and specificity of UroVysion FISH to be 72% and 83% for the detection of UC, compared to 42% and 96% for cytology, respectively.8
Accordingly, a more recent study found a significant difference between the overall sensitivities of FISH and cytology (60% versus 28.4%, respectively; p<0.0001) and more interestingly, the severity of the genetic alterations detected by FISH showed a positive correlation with both tumour invasiveness (stage Ta/T1, T2) and histological grade (G1, G2/G3).9
FISH test for upper tract urothelial carcinoma
Because of its high reliability and the proven molecular similarity of urothelial cancers of the bladder and the upper urinary tract (UUT), the clinical utility of this FISH assay has been also evaluated for diagnosing and monitoring of the UUT UC, in both voided urine and endoscopically collected samples of ureteral washing and urine.
The diagnostic value of FISH for UUT tumours was shown in studies from voided urine in patients with known urothelial cancers in comparison with healthy persons and with patients with benign urological diseases.
In those studies, reported sensitivities and specificities range from 73.5–85.7% and 94–100%, respectively. FISH was, furthermore, evaluated in UUT washing urine of patients with suspected UUT tumours: this analysis revealed the highest sensitivities (100%), probably because of the direct access to mobilised urothelial cells.10–12 However, FISH is not approved as a tool for UUT tumours and studies evaluating this test in the UUT UC remain scarce.
Our Institution experience
At our institution, in collaboration with the San Raffaele Urology Deparment, the urine FISH assay was set up with a successful 30-case pilot study between 2008 and 2009, and it has been offered to patients and clinicians as routine test since 2010.
In particular between 2010 and 2015 a total of 480 samples were submitted to the Molecular Oncologic Diagnostic Laboratory of the San Raffaele Hospital Pathology Unit for urine FISH analysis: 402 samples of voided urine and 78 samples of upper tract/bladder washes; 30–50ml of spontaneous urine and 5–20ml of endoscopical bladder and UUT washes were collected in sterile containers with no additional fixative and processed within two hours, following classical cytogenetics protocols (hypotonic treatment and methanol: acetic acid, 3:1 fixation).
Approximately 20µl of the final cell suspension was dropped onto a standard glass slide, which was air-dried and hybridised, according to FISH probes manufacturer indication. The samples were all tested within two days of the sample processing, using the UroVysion kit, Abbott Molecular (between 2010 and 2012), and the ZytoLight SPEC p16/CEN 3/7/17 Quadruple Color Probe, Zytovision (between 2012 and 2015), with high quality comparable results.
Two independent observers, both trained cytogeneticists with experience in cytology, evaluated all of the samples, looking for at least 25 morphologically suspicious nuclei and all the observed FISH signals were recorded for each nucleus. Correlation with urine cytology and/or bladder histology was made, if data were available.
Of the total 480 samples, 275 were submitted for surveillance (surveillance group) and 148 for haematuria/diagnosis (diagnosis group); the remaining 57 samples had no clinical indication specified on the request. All the test requests came from either the urologist/oncologist or the family doctor.
Following the UroVysion scoring criteria, 152 were positive (88 were submitted for haematuria/diagnosis and 64 were for surveillance), 315 were negative (201 for surveillance and 114 for diagnosis) and 13 were not diagnostic (no cells/no urothelial cells).
Interestingly 33.5% of the FISH positive cases (51 out of 152 – 20 of the surveillance group and 31 of the diagnosis group) had a paired negative urine cytology/bladder bioptic histology.
Conclusions
In line with the above-mentioned literature, we observed that our results showed an increased sensitivity in detecting neoplastic lesions, which were proven to be true with successive clinical analysis (cystoscopy, CAT scans) and/or bioptic samplings.
In fact, several follow-up studies reported that almost half of the patients with initial positive FISH tests and negative cystoscopy results experienced disease recurrence within the year after the test, suggesting that the detection of chromosomal abnormalities anticipated the diagnostic of recurrence by cystoscopy or urinary cytology and accordingly, also in our experience an abnormal urine FISH test can constitute an accurate surveillance assay by anticipating disease recurrence.13
Some other studies promoted a reflex FISH in cases of atypical cystoscopy or cytology, in order to assess both tumour recurrence and new diagnosis, and interestingly, this type of strategy may reduce the number of unnecessary biopsies and may be cost effective.14,15
Because of some FISH disadvantages, including its reagents and instrumentations costs and the need for experienced personnel, the clinical use of FISH remains lower than expected.
Nevertheless our six-year activity, which still needs to be expanded and analysed more accurately, suggests the usefulness of such a test as a non-invasive UC diagnostic and prognostic tool.
References
- Phillips JL, Richardson IC. Aneuploidy in bladder cancers: the utility of fluorescent in situ hybridization in clinical practice. BJU Int 2006;98(1):33–7.
- Krause FS et al. Clinical decisions for treatment of different staged bladder cancer based on multitarget fluorescence in situ hybridization assays? World J Urol 2006;24(4):418–22.
- Tsuchiya KD. Fluorescence in situ hybridization. Clin Lab Med 2011;31(4):525–42.
- Inoue T et al. Chromosomal numerical aberrations of exfoliated cells in the urine detected by fluorescence in situ hybridization: Clinical implication for the detection of bladder cancer. Urol Res 2000;28:57–61.
- Sokolova IA 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.
- Bubendorf L et al. Multiprobe FISH for enhanced detection of bladder cancer in voided urine specimens and bladder washings. Am J Clin Pathol 2001;116:79–86.
- Mbeutcha A et al. Current status of urinary biomarkers for detection and surveillance of bladder cancer. Urol Clin North Am 2016;43(1):47–62.
- Hajdinjak T. UroVysion FISH test for detecting urothelial cancers: Meta-analysis of diagnostic accuracy and comparison with urinary cytology testing. Urol Oncol 2008;26:646–51.
- Song MJ, Lee HM, Kim SH. Clinical usefulness of fluorescence in situ hybridization for diagnosis and surveillance of bladder cancer. Cancer Genet Cytogenet 2010;198(2):144–50.
- Fadl-Elmula I et al. Cytogenetic analysis of upper urinary tract transitional cell carcinomas. Cancer Genet Cytogenet 1999;115(2):123–7.
- Luo B et al. Utility of fluorescence in situ hybridization in the diagnosis of upper urinary tract urothelial carcinoma. Cancer Genet Cytogenet 2009;189(2):93–7.
- Gruschwitz T et al. FISH analysis of washing urine from the upper urinary tract for the detection of urothelial cancers. Int Urol Nephrol 2014;46(9):1769–74.
- Seideman C et al. Multicenter evaluation of the role of UroVysion FISH assay in surveillance of patients with bladder cancer: does FISH positivity anticipate recurrence? World J Urol 2015;33(9):1309–13.
- Schlomer BJ et al. Prospective validation of the clinical usefulness of reflex fluorescence in situ hybridization assay in patients with atypical cytology for the detection of urothelial carcinoma of the bladder. J Urol 2010;183:62–7.
- Kim PH et al. Reflex fluorescence in situ hybridization assay for suspicious urinary cytology in patients with bladder cancer with negative surveillance cystoscopy. BJU Int 2014;114:354–9.
