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Urothelial carcinoma accounts for the overwhelming majority of bladder cancers and has various clinical manifestations with different prognostic and therapeutic implications. Because of the high recurrence risk (up to 70%) and up to 30% with stage and grade progression, the long-term follow-up of such patients is mandatory [1,2].
Currently, the combined use of cystoscopy and urine cytology is the standard approach for the diagnosis and surveillance of bladder cancer. However, cystoscopy is invasive, expensive and has poor sensitivity in detecting flat urothelial lesions, including carcinoma in situ. In contrast, voided urine cytology is noninvasive, convenient and easy to administer. It is an excellent tool for detecting high-grade tumours, with a sensitivity as high as 95% and a specificity of up to 100%. The major disadvantage of the method is its low sensitivity for low-grade urothelial tumours (15–20%) . One of the most important variables affecting the overall sensitivity and specificity of urinary cytology is specimen quality, which can be significantly improved by using liquid-based thin-layer technology (e.g. ThinPrep®, CYTYC Corp., Boxborough, MA, USA) .
Regardless of their indisputable diagnostic value, neither test alone or combined is sufficient for the early detection, assessment of recurrence or progression of bladder cancer. Therefore, numerous ancillary noninvasive molecular tests are being actively investigated for improving the sensitivity of cytology and to avoid unnecessary cystoscopies, particularly in patients with low-risk disease .
Adjuvant fluorescence in situ hybridization (FISH) has been confirmed as a promising tool for monitoring recurrent bladder cancer, providing better sensitivity than standard urine cytology while maintaining its high specificity . The commercial multitarget, multicolor UroVysion FISH assay (Vysis-Abbott, Downers Grove, IL), which was designed to detect aneusomy of chromosomes 3, 7 and 17, and loss of the 9p21 locus that occur frequently in bladder cancer, has recently made the FISH technique available in routine cytopathology laboratories [8,9].
In the present pilot study, we aimed to determine the practical utility of the UroVysion FISH assay for detecting the presence and predicting the likelihood of bladder cancer recurrence/progression, based on the analysis of one voided urine cytology specimen (ThinPrep) used for routine diagnosis. The specific goal was to determine whether it is possible to distinguish between patients with ‘low-risk’ and ‘high-risk’ superficial bladder cancer based on the chromosomal pattern detected by FISH in follow-up cytology, using optimized methods for molecular cytology and signal evaluation.
PATIENTS AND METHODS
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- PATIENTS AND METHODS
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Urinary cytology samples from 47 consecutive patients (41 men and six women; mean age 69 years, range 28–88) with urinary tract neoplasms were assessed retrospectively; 34 were under surveillance for recurrence of superficial bladder cancer and benign papillary neoplasms after complete transurethral resection, with a mean follow-up of 29 (range 6–139) months, and 13 had no history of urinary tract malignancy. Voided urine specimens and concurrent or follow-up biopsies were obtained from different outpatient urological practices and hospitals from the federal state North Rhine-Westphalia, West Germany. Any cystoscopically suspicious lesion was biopsied or removed transurethrally. Tumour classification was in accordance with guidelines of the WHO  and Union Internationale Contre le Cancer . Histological diagnoses of primary bladder tumours included urothelial carcinoma of grades 1 (12), 2 (16) and 3 (eight), and stages pTa in 19, pTis in two, pT1 in 12, and pT3 and pT4 in one each; urothelial dysplasia grade 2 was diagnosed in one, papillary urothelial neoplasm of low malignant potential (PUNLMP) and papilloma in two patients each. Extravesical locations of malignancies included the ureter (in three; once as a metastasis from a primary sigma adenocarcinoma), the renal pelvis (in two), the kidney and the prostate (one each).
Since 1999, all urine samples have been prepared by the liquid-based cytology technique (ThinPrep) at our institute and we supply all clinical laboratories with ThinPrep ‘Extragyn’ collection vials containing ‘Preserve Cyt’ solution for enabling immediate fixation of urine. Urine was collected at the referring urological practices after provoking a high urine output by recommending the patients to drink copious amounts water or tea 1.5–2 h before visiting the practice. About 50 mL of each probe of clean-catch midstream urine was immediately sedimented at 827 g for 10 min; the cell pellet was re-suspended in 10 mL methanol-based fixative (Preserve Cyt) and sent to our institute for further processing. Cytology slides were prepared in the ThinPrep processor and stained according to Papanicolaou. All cytology diagnoses were originally made and re-reviewed for the present study by an experienced cytopathologist (M.B.). Cytology diagnoses included ‘positive’, ‘suspicious’ and ‘negative’ for malignancy; equivalent results were considered as ‘positive cytology’ for statistical analysis. The location of all cytologically abnormal transitional cells was labelled on the coverslips using a permanent marker to facilitate subsequent FISH analysis.
The UroVysion FISH test was applied to the same ThinPrep slide used for diagnosis. During monitoring, one FISH test was performed either on the first abnormal cytology monolayer, or once by chance in patients with a long uneventful clinical course and negative morphology. The laboratory procedure was according to the instructions of the manufacturer. Briefly, after removing coverslips in xylene, slides were rehydrated through two series of 100%, 95% and 70% ethanol, placed under running tap water for 5 min, and then in 0.5% HCl in 70% ethanol for 15 min. After immersion in 2 × saline sodium citrate (SSC) for 5 min at 73 °C, slides were briefly air-dried and digested by using 0.2 mg/mL pepsin in 0.01 mol/L HCl for 15 min at 37 °C in a humidified chamber. Subsequently, slides were washed in PBS for 5 min at room temperature, fixed in 1% neutral buffered formalin/PBS for 5 min, washed in PBS for 5 min again and air-dried. 10 µL of the hybridization mix (8 µL hybridization buffer LSI-WCP, Vysis-Abbott, 1 µL distilled water and 1 µL Urovysion probe mix), consisting of directly labelled fluorescent probes to the pericentromeric regions of chromosomes 3 (CEP-3-red), 7 (CEP-7-green) and 17 (CEP 17-aqua) and to the locus 9p21 (LSI 9p21-gold), was placed on the target, cover-slipped and sealed with rubber cement. After co-denaturation at 73 °C for 10 min, slides were incubated at 37 °C overnight in a humidified chamber. After hybridization the samples were washed in 2 × SSC at 73 °C and in 2 × SSC at room temperature for 2 min each. Then 4,6-diamidine-2-phenylindole dihydrochloride was used for counterstaining.
Each case was evaluated while unaware of the clinical or cytology findings by an independent observer (H.H.) experienced with FISH analysis. Slides were scored for hybridization signals using a Axioplan fluorescence microscope (Zeiss, Jena, Germany) with an appropriate filter set at × 400. Spots were counted primarily in previously marked areas by automatic interphase FISH analysis using the Metafer-Metacyte slide scanning system (Metasystems GmbH, Altlussheim, Germany) (Fig. 1a). At least 30 abnormal nuclei per slide were evaluated in each case (Fig. 1b,d), and normal-appearing urothelial cells in the same monolayer were analysed for internal control. FISH results were considered positive if: (i) ≥ 10 cells had deletion of 9p21 (homozygous or heterozygous); (ii) if ≥ 10 cells had polysomy of one chromosome; or (iii) if ≥5 cells had polysomy of more than one chromosome per cell (Vysis-Abbott Diagnostics). Tetraploid chromosomal sets were not considered as a sign of malignancy.
Figure 1. a, Characteristic panel of a Metafer-MetaCyte gallery with images of selected atypical nuclei after multicolour, multiprobe FISH. b, Example of mildly atypical transitional cells from a noninvasive, low-grade papillary urothelial carcinoma in a Papanicolaou-stained ThinPrep monolayer. c, Concurrent UroVysion FISH result showing ‘low-risk FISH pattern’ with heterozygous deletion of 9p21 (one gold signal) and disomy of chromosomes 3 (red), 7 (green) and 17 (aquamarine). d, Example of a markedly enlarged, hyperchromatic nucleus of a highly atypical transitional cell with coarse chromatin and prominent nucleoli in a ThinPrep monolayer stained according to Papanicolaou. e, Concurrent UroVysion FISH results showing ‘high-risk FISH pattern’ with heterozygous deletion of 9p21 (one gold signal) and different polysomies of chromosomes 3 (four red signals), 7 (four green signals) and 17 (three aquamarine signals)
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The mean copy number of each chromosome (the chromosome index, CI, the total number of hybridization signals divided by the total number of nuclei analysed) and the frequency of polysomies (polysomy index, PI, the percentage of scored nuclei with ≥ 3 chromosome copies) were calculated to estimate the degree of chromosome instability in each case.
FISH results were classified into three groups for prognostic analysis: (i) negative FISH, with a disomic chromosomal pattern; (ii) ‘low-risk FISH’, showing loss of 9p21 either as the only chromosomal aberration or accompanied by single cells (<10%) with tri- or tetrasomy/ies of either or all chromosomes 3, 7, and 17 (Fig. 1c); and (iii) ‘high-risk FISH’, with loss of 9p21 and > 10% aneusomy/ies for either or all marker chromosomes (Fig. 1e). For statistical purposes, negative FISH results were grouped together with low-risk cases. For assessing clinical outcome, the following categories were used: (i) uneventful, no signs of disease recurrence or progression during follow-up; (ii) recurrence, reappearance of neoplasia after therapy in the same stage and grade as the primary tumour; and (iii) progression, disease reappearance in advanced stage and/or grade.
Descriptive statistics for continuous measures are given as the mean and/or median and range. Pearson's chi-square test was used to compare chromosomal patterns with clinical and pathological findings. Correlations between variables were analysed using Spearman's rank correlation. Differences in the clinical outcome between patient groups with different chromosomal patterns were evaluated by the Mann–Whitney U-test.
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Of the 47 patients, 13 had no history of urothelial malignancy; the tumour characteristics and test results are shown in Table 1. Three of six bladder carcinomas were not detected by cystoscopy (three of six false-negative). In contrast, all six tumours were positive both by cytology (three positive, two suspicious and one equivocal) and FISH. The remaining seven tumours were extravesical; none of them was detected by cystoscopy, as expected, whereas all were positive by FISH (9p21 loss plus various aneusomies of chromosomes 3, 7 and 17). All tumours but one ureteric carcinoma were also positive by cytology. This shows that all urothelial malignancies were correctly detected as true positives by the UroVysion FISH test (no false negatives), and all but one were also defined as true positives using cytology (one of 13 false-negative). To avoid statistical invalidity because of the few samples assessed in the present pilot study, further test performance characteristics were not calculated.
Table 1. Tumour characteristics and test results in 13 newly diagnosed malignancies in the urinary tract
|Patient||Material||Cytology diagnosis||UroVysion FISH||Cystoscopy||Histology||Location|
|11||VU||equivocal||+||−ve||UC pT3pN2||renal pelvis|
| 8||UW||suspicious||+||−ve||UC pTaG2||renal pelvis|
| 6||VU||suspicious||+||−ve||RCC pT1a pN1||kidney|
Thirty-four of the patients were under surveillance for recurrence of superficial bladder neoplasia. The relationship between primary tumour characteristics and chromosomal pattern by FISH in follow-up urine cytology is shown in Table 2. Statistical analysis showed a highly significant correlation between malignancy grade and stage of the primary tumours and chromosomal instability by FISH in follow-up urine cytology (rs− 0.583, P < 0.001, and −0.451, P = 0.004, respectively; Spearman's rank correlation).
Table 2. Relationship between primary tumour characteristics and chromosomal pattern in follow-up urine cytology in 34 bladder neoplasms
|Primary histology and stage, if appropriate, and grade||FISH|
|urothelial papilloma||1|| 1||–|
|flat urothelial dysplasia/G2||–|| –||1|
The UroVysion test was negative in four, showed a low-risk pattern in 17 and a high-risk pattern in 13 patients. The selection, classification and outcome of the patients are shown in Fig. 2. During a mean follow-up of 29 (range 6–139) months, one papilloma of the four FISH-negative tumours, four neoplasms of the 17 low-risk FISH cases (one PUNLMP, two pTaG1, one pT1G1) and five carcinomas of 11 high-risk FISH-positive patients developed histologically verified recurrence with no disease progression.
Figure 2. The selection, classification and outcome of 47 patients with biopsy-confirmed urinary tract neoplasia (UN) based on the chromosomal pattern detected by the UroVysion FISH assay. *Anticipatory positive FISH is no biopsy-confirmed advance of disease until the closing date of the study, but the latest FISH assays showed high-frequency polysomy of all chromosomes investigated.
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Intriguingly, there was recurrence in the low-risk FISH group only in patients with 9p21 loss and additional trisomies and/or tetrasomies of marker chromosomes in single cells; there were no recurrences in patients with 9p21 deletion only (five of 17; mean follow-up 41 months, range 6–98). The disease progressed only in patients with high-risk FISH (four of 11), in whom FISH findings preceded by a mean of 5.5 (1–12) months the cystoscopic and histological verification. In two patients, no final histological result of disease recurrence/progression was available by the closing date of the study, but the latest FISH assays during monitoring revealed high-risk chromosomal aberrations (anticipatory positive result).
Two patients with high-risk FISH findings (histologically dysplasia G2 and PUNLMP) had no disease recurrence or progression during the follow-up (17 and 24 months, respectively). Both tumours were at the lowest end of the high-risk FISH spectrum, containing low-frequency trisomies and tetrasomies of marker chromosomes beside the moderate rate of p921 loss.
Statistical analysis showed significant differences both in recurrence rate (P = 0.026; Mann–Whitney) and progression rate (P = 0.015; Mann–Whitney) between groups with low-risk and high-risk FISH results.
In general, a numerical aberration of chromosome 3 occurred in 21/34 patients (62%, CI 3–4.9), chromosome 7 in 24/34 (70%, CI 3–5.6), chromosome 17 in 19/34 (56%, CI 3–5.2), and loss of 9p21 in 88% (median 37%, range 13–100) in all bladder tumours investigated. As shown in Table 3, the CIs of chromosomes 3, 7 and 17 were equal or very similar between tumours with recurrence only and those with progression. In contrast, there were statistically very significant difference between the CIs of chromosome 3, 7 and 17 in tumours with an uneventful clinical outcome compared with those which developed recurrence and/or progression (P < 0.01 for each by the Mann–Whitney test). However, progressive tumours had a significantly higher frequency of polysomies in chromosomes 3, 7 and 17 (median 55%, 77% and 63%, respectively) compared with values in recurrent diseases (27%, 11% and 18%, respectively; P < 0.01 for each, Mann–Whitney). Intriguingly, there was no significant difference in the median frequency of 9p21 loss between prognostic groups.
Table 3. Relationship between overall CI and PI in groups of patients with bladder neoplasia with a different clinical outcome
|Median (range) CI (n) or PI (%)||Clinical course|
|Uneventful||Recurrence only||Recurrence + progression|
|3 CI|| 2 (2–4)|| 3.6 (2–4.1)‡|| 3.6 (3.4–4.9)†|
|3 PI|| 0 (0–40)||27 (0–75)‡||55 (45–86)†|
|7 CI|| 3 (2–4)|| 3.4 (2–3.7)†|| 3.4 (3.4–5.6)†|
|7 PI|| 4 (0–33)||11 (0–61)*||77 (72–100)†|
|17 CI|| 2 (2–4)|| 3.25 (2–4)†|| 3.4 (3.3–4.6)*|
|17 PI|| 0 (0–25)||18 (0–61)‡||63 (50–95)†|
|Rate of 9p21 loss||28 (0–50)||33 (0–80)||23 (22–100)|
Surveillance cystoscopy was positive in four of 10 patients with recurrent disease, but otherwise negative in all others with a long-term uneventful clinical outcome, and in patients with progressive tumours. Urine cytology detected eight of 10 patients with recurrent tumours and all four with progressive disease. FISH detected nine of 10 recurrent tumours (rs− 0.417, P = 0.018) and all four patients with progressive disease (rs − 0.404, P = 0.022). All progressive tumours were characterized by a high-risk type chromosomal pattern by UroVysion, as expressed by 9p21 deletion plus particularly high frequency polysomies of chromosomes 3, 7 and 17.
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Newer protocols for treatment and surveillance stratify patients with superficial bladder cancer into low-risk and high-risk groups for disease recurrence and progression, on the basis of initial stage and grade of the tumour . However, it is not possible individually to predict the clinical course of bladder cancer by cystoscopy and pathology alone. Interphase FISH is increasingly used as an adjunct for detecting and monitoring urothelial malignancies. The UroVysion FISH assay enhances the sensitivity of conventional urinary cytology [8,9] and helps in clarifying ambiguous cytology results .
In the present study, no overt malignant tumour in the urinary tract was undetected by the UroVysion test, and only one of 13 was false-negative by cytology of voided urine and ureter-washing samples. Respective results from previous larger studies indicate a 19–27% false-negative rate for FISH and 32–42% for cytology [7,8,14]. This might be partly explained by our use of UroVysion on liquid-based ThinPrep urinary cytology preparations, which improves cell yield and cell preservation, with a significant reduction of background artefacts , and markedly enhances the sensitivity of ancillary UroVysion FISH test by up to 96–100%[15,16]. Furthermore, the use of automated interphase ‘spot counting’ by the Metafer-Metacyte system in the present study reportedly increases the sensitivity and efficiency of signal enumeration, especially in rare cells, if compared with manual counting .
In the present study cystoscopy was positive in three of six primary bladder tumours and negative in all extravesical malignancies, as expected. This shows that UroVysion may detect cancer cells in the urine before tumour growth can be identified by visual inspection, or by microscopy, indicating that early genetic changes do not necessarily correlate with observable changes in morphology.
Another important feature of FISH has become apparent on analysing tumour behaviour in association with the pattern of chromosomal aberrations. During a mean follow-up of 29 months, five of the 21 patients with negative- and low-risk FISH results developed recurrence. None of the patients with only 9p21 deletion on follow-up cytology had tumour recurrence or progression during a mean follow-up of 41 (6–98) months. This indicates that these tumours have a relatively indolent clinical outcome, with long disease-free intervals and low recurrence or progression rate, if at all. In patients of the low-risk FISH group, there were recurrences only in those whose cytology sample contained single-cell trisomies and/or tetrasomies (<10%) of any of chromosomes 3, 7 and 17, in addition to deletion of 9p21. This is consistent with findings of other studies suggesting that in tumours in which loss of chromosome 9 sequences is the only chromosomal change, there appears to be little tendency towards aggressive behaviour . Similar results were reported by Pycha et al., who found that superficial, intermediate-risk tumours of patients with negative FISH or those with only 9p21 and/or chromosome 3 irregularities developed recurrences in 15% of cases.
By contrast, nine of the 11 patients with a high-risk FISH pattern had recurrence and five of them had tumour stage/grade progression during the follow-up. This supports previous results of others reporting that aneusomy of chromosomes 7 and 17 in primary tumours are potential risk factors for bladder cancer recurrence [20,21] and additional genetic imbalances, especially at 3p, 11p, 17p, and 18q chromosomal arms, arise as superficial tumours progress into muscle-invasive carcinomas .
The present findings of a highly significant direct correlation between malignancy grade and stage of the primary tumours and the level of chromosomal instability by FISH in follow-up urine cytology is concordant with results from several groups, indicating that tumour progression is associated with the accumulation of genetic abnormalities [1,2,18]. The present results also confirm previous findings on the utility of the FISH assay in identifying more aggressive types of bladder cancer [19,21,23].
The phenomenon that chromosomal aberrations in urinary cytology by FISH precede visible or histologically detectable tumour by several months was described in all larger studies using UroVysion [7,8,13,24,25]. In general, UroVysion detects bladder cancer recurrence or progression up to 6 months earlier than other diagnostic methods (anticipatory positive results by FISH). This indicates that genetic/chromosomal aberrations in urothelial cells may persist for several months after the primary bladder cancer had been removed, as a hallmark of generalized, ongoing genetic instability in the whole tissue field of carcinogen-exposed bladder mucosa, as reported by others . In the present retrospective study, four patients were identified with such a disease course. The mean follow-up after positive FISH until histological verification was 5.5 (1–12) months.
The present results show (Table 3) that the mean signal counts (CI) for chromosomes 3, 7 and 17 were significantly higher in patients with recurrent and progressive tumours than in those with an uneventful clinical outcome; the association was also similar for polysomy frequencies (PI). However, the CIs were similar between groups with low-risk and high-risk disease, showing a significant overlap between cases in different risk categories. Nor was there a significant difference in the frequency of 9p21 loss between groups of tumours with different clinical outcome. True ‘high-polysomy’ (a mean of five or more signals in ≥ 2% of cells , which was found for chromosomes 7, 8, 9, and 17, and significantly associated with tumour progression [28–30], was noted only once for chromosome 7 in the present study in a patient with progressive disease.
Intriguingly, the only variable with a statistically significant difference between patient groups with only recurrent tumour or progressive disease was the frequency of polysomy (PI) for chromosomes 3, 7 and 17. In particular, the median (range) PI of progressive tumours was significantly higher than that for recurrent low-risk disease. As high-frequency polysomies always affected more than one chromosome, we presume that these changes reflect the overall genetic instability of bladder mucosa rather than specific genetic alterations during tumour development, and are therefore suggestive of aneuploidy.
We conclude that the combination of UroVysion FISH with ThinPrep technology and automated signal counting (Metafer-Metacyte system) provides significantly better diagnostic tools in urinary tract malignancies. Our preliminary data strongly suggest that it is possible to predict the clinical behaviour of superficial bladder cancer by using the UroVysion FISH test on scheduled follow-up urinary cytology preparations. Based on the chromosomal pattern, patients with an anticipated uneventful clinical course and/or recurrent low-grade papillary tumours can be distinguished from those who are at a high risk of tumour progression. Accordingly, the timing of follow-up cystoscopies could be tailored for individual patients on the basis of the biological properties of their disease. Further larger prospective studies are required to confirm the usefulness of this approach before it can be definitively incorporated into the clinical setting.