Article Text
Abstract
Aim: To establish independent prognostic factors on a chromosomal basis in superficial bladder cancer, using a multicolour fluorescence in situ hybridisation (FISH) probe mix.
Patients and methods: In 2002, voided urine from 75 consecutive patients (mean age 71.7, range 52–93) years under follow-up for superficial urothelial cancer was studied prospectively. The patients were observed for a mean (standard deviation (SD)) period of 39.3 (6.8) months (range 27–58) until July 2005. A multicolour FISH on liquid-based voided urinary cytology was carried out on all patients. Univariate analysis, using a log rank test, was used to determine the prognostic relevance of a low-risk pattern and a high-risk pattern. Progression-free survival time was calculated from the date of first diagnosis to first recurrence or progression according to the Kaplan–Meier product-limit method.
Results: One patient was lost to follow-up. 27 of the 74 remaining (36.8%) patients showed recurrent disease. In 9 (33.3%) patients with a low-risk pattern disease recurred after a mean (SD) observation time of 29.7 (1.9) months (range 8.3–52.3, median 30.8 (12.4)). 18 (66.7%) patients with a high-risk pattern developed recurrence within a mean (SD) of 17.6 (2.0) months (range 4–38.8, median 16.7 (11.6)). The Kaplan–Meier curve for progression-free survival showed marked differences between the low-risk and the high-risk groups.
Conclusion: Patients with a high-risk chromosomal pattern have a markedly shorter disease-free survival time and higher progression rate than patients with a low-risk pattern. High-risk patients can therefore be treated more aggressively to prevent tumour spreading.
- Cis, carcinoma in situ
- FISH, fluorescence in situ hybridisation
- SSC, sodium saline citrate
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At diagnosis, most cases of urothelial cancer are superficial. About 40% are papillary pTa urothelial cancer, 30% pT1 urothelial cancer and 2–5% are carcinoma in situ (Cis).1,2 But this is not a homogeneous entity. Oncogenesis of pTa and pT1–Cis runs along different pathways,3 with different oncological potentials and, therefore, varying clinical outcomes. Loss of heterozygosity of chromosomes 9p and 9q has been shown to be a crucial event in the transition of normal urothelium to papillary urothelial carcinoma, and p53 is primarily associated with the development of Cis.4 Several studies described pTa urothelial carcinomas as having less of a propensity to progress4,5 than pT1 tumours, which reach a progression rate of between 30% and 50%.4–7 Tumour grade and stage are established prognostic factors in bladder cancer.6–8 The problems of endoscopic assessment of urothelial carcinoma are well known. Tumour localisation, specimen fulguration at transurethral resection, cutting artefacts during slide preparation and depth of the cutting strip are all obstacles to correct histological evaluation. Comparative investigations of the same slides in different pathological institutions produce varying results.
They are, however, dependent on the investigator, and on morphometric and morphological parameters. The tumour grade, especially, underlies a high interobserver variability and there are shortcomings with regard to unbiased evaluation.9 Little progress, if any, has been achieved in the past 30 years. Therefore, there is a need for additional parameters that allow a more accurate assessment of the biological behaviour of the tumour.
Since its discovery as an inhibitor of cyclin-dependent kinases 4 and 6, the tumour suppressor p16 has attracted much attention in cancer research. Loss of p16 in carcinogenesis is an early and often critical event in the progression of many tumours.10 Loss of one or both alleles of the p16 gene in human papillary urothelial cancer has been suggested to play a major part in early carcinogenesis.11,12 Previous studies have reported an important correlation between p16 immunoreactivity13,14 and loss of p16 expression and tumour progression in patients with minimally invasive urothelial cancer. Other immunohistochemical parameters, such as p53 antigen expression,14,15 and other cell cycle proteins14,16 as well as p16 expression were also described.
Interphase fluorescence in situ hybridisation (FISH) is increasingly used as an adjunct method in the diagnosis of malignancies, such as in urothelial cancer. Using a multicolour, multitarget interphase FISH probe mix containing centromere probes for chromosomes 3, 7 and 17 and a locus-specific probe to the band 9p21 (p16), we tried to characterise the biological behaviour of urothelial cancer and to establish independent prognostic factors on a chromosomal basis.
PATIENTS AND METHODS
In 2002, voided urine from 75 consecutive patients under follow-up after transurethral resection of urothelial cancer (mean age 71.7, range 52–93) years was studied prospectively. The patients were followed for a mean (standard deviation (SD)) observation time of 39.3 (6.8) months (range 27–58) until July 2005. In all patients a multicolour FISH was performed on liquid-based voided urinary cytology. During follow-up, any cystoscopically suspicious lesion was examined or removed transurethrally. Histopathological classification was carried out according to Union Internationale Contre le Cancer criteria.17 Table 1 gives the histopathological diagnoses.
Fresh voided urine was added to previously prepared Falcon tubes containing 15 ml Cytolyt (CYTYC Corp, Boxborough, Massachussetts, USA) and sent to the laboratory. After centrifugation for 10 min at 2000 rpm, the cell pellet was resuspended in a ThinPrep extragyn phial (CYTYC Corp) containing the Preservcyt solution (CYTYC Corp). All specimens were prepared on Superfrost plus slides using the ThinPrepSystem (CYTYC Corp). Cells were fixed using a 50% isopropanol spray.
Multicolour FISH
After pretreatment with 2× sodium saline citrate (SSC) and 0.5 mg/ml pepsin at 37°C and 0.01 N hydrochloric acid in a waterbath, 1% formaldehyde and phosphate-buffered saline at room temperature, the slides were dehydrated in 70%, 80% and 100% ethanol. After drying the slides, the probe mix (Vysis, Downers, Illinois, USA) containing a locus-specific probe for the locus 9p21 (p16) and three α-satellite-bound centromere-specific probes for chromosomes 3, 7 and 17 was placed on the target. Codenaturation (5 min at 73°C) and hybridisation at 37°C were carried out overnight in the HYBrite oven (Vysis). The procedure was followed by a post-wash using 0.4× SSC and 0.3% Nonidet (NP40) and 2× SSC and 0.1% NP40. Diamidinophenylindole II was used as a counterstain.
Slides were scored for hybridisation signals on a cell-by-cell basis, using an Olympus Provis AX 70 (Olympus, Italy) with a filter set including diamidinophenylindole single bandpass (counterstain), aqua single bandpass (chromosome 17), yellow single bandpass (9p21 locus) and a red–green double bandpass (chromosomes 3 and 7). Enumeration and evaluation of the FISH signals was carried out on target cells that appeared abnormal morphologically, according to Bubendorf et al,18 and the cut-off level was set at ⩾4 aneusomic cells.
According to a previous study,19 patients with a diploid chromosomal pattern or only p16 or chromosome 3 positivity were considered to be at low risk for recurrence or progression, whereas patients with a chromosomal pattern including aberrations of chromosomes 7 or 17 were considered to be at high risk.
Statistical evaluation
Univariate analysis using the Mantel–Cox log rank test was used to determine the prognostic relevance of diploid pattern, p16 or chromosome 3 positivity (low risk) and chromosome 7 or 17 positivity (high risk). A p value <0.05 was considered to be significant. Disease or progression-free survival time was calculated as the period between the date of first diagnosis and first recurrence according to the Kaplan–Meier product-limit method.20
RESULTS
On multicolour FISH analysis, 17 of the 75 patients were negative (diploid) and 58 patients were positive (aberration of any analysed probe), with 25 of the 58 FISH-positive patients showing loss of one or both alleles of p16 or chromosome 3 positivity. The low-risk group consisted of 42 (56.0%) patients with a diploid chromosomal pattern or only p16 or chromosome 3 positivity. In all, 33 of 75 (44.0%) patients who had aneuploidy of chromosome 7 or 17 formed the high-risk group (table 2). One patient was lost to follow-up. The mean (SD) observation time of the remaining 74 patients was 39.3 (6.8) months (range 27–58).
In total, the disease recurred in 27 of 74 (36.8%) patients after a mean (SD) observation time of 24.5 (1.5) months (range 4–52, median 26.6 (13.4)). In the low-risk group, 4 of the 17 (23.5%) FISH-negative patients had histologically verified recurrence and 5 of 25 (20.0%) patients with p16 or chromosome 3 positivity showed recurrent disease. One (11.1%) patient in this group showed progression from stage pTaG1 to stage pTaG3 after 36 months. In the high-risk group, disease recurred in 18 of 33 (54.5%) patients (table 3). The progression rate in this group was 50.0% (9/18).
In 9 (21.4%) patients with a low-risk chromosomal pattern disease recurred after a mean (SD) observation time of 29.7 (1.9) months (range 8.3–52.3, median 30.8 (12.4)). The 18 patients (54.5%) with a high-risk pattern showed recurrence within a mean (SD) of 17.6 (2.0) months (range 4–38.8, median 16.7 (11.6)). The Kaplan–Meier curve for disease or progression-free survival showed marked differences between the low-risk and the high-risk groups (fig 1).
DISCUSSION
Many studies have shown that the prognosis for recurrence, progression and survival is considerably worse for patients with pT1 than for patients with pTa tumours. In addition, multivariate analyses of these studies identified several risk factors such as stage, grade, multifocality, tumour size and results at 3-month cystoscopy. Invasion of the lamina propria seems to be the most important indicator for progression. However, the natural history of pTa tumours differs so vastly from pT1 tumours that some authors21 distinguish between pTa tumours and pT1 tumours.
As mentioned before, the evaluation of bladder cancer is fraught with problems of objectivity. The interobserver bias regarding the tumour grade is particularly high and often influences the decision-making process.9 Today, the flaws in bladder cancer staging are nearly the same as 30 years ago. Therefore, there is an additional need for objective parameters allowing the more accurate assessment of the tumour’s biological behaviour. In this way, patients at high risk for tumour progression can be treated more aggressively to prevent tumour spread of and metastasis; low-risk patients, on the other hand, may be spared aggressive treatment and be followed up at longer intervals.6,8,19 Furthermore, new prognostic parameters could provide additional arguments for therapeutic decisions in those cases in which conventional prognostic parameters point to divergent prognostic outcomes.6
Mutation of cell-cycle regulatory genes is the genetic change most commonly found in human neoplasia, including bladder cancer.22,23 Recently, a new family of negative cell-cycle regulators, the cyclin-dependent kinase inhibitor genes INK4 and KIP, has been identified.24 Loss of p16 expression, a protein encoded by the INK4 family member INK4A, has been associated with hyperphosphorylation of the retinoblastoma gene. Several studies have reported its prognostic relevance (with p53, pRB and p21) in patients treated with radical cystectomy for bladder cancer.6,14
Interphase FISH is increasingly used as an adjunct method in the diagnosis of malignancies. As discussed previously,19 the multicolour-FISH technique can be used to classify patients at different levels of risk of recurrence or progression, to distinguish patients with a “benign” chromosomal pattern, who can be followed up like low-risk patients, from patients with a more aggressive chromosomal pattern who have to be followed up like high-risk patients. In fact, in our study, the Kaplan–Meier curve for progression-free survival showed a highly marked difference between the two groups of patients with low-risk and high-risk patterns. Of the low-risk group, 21.4% showed recurrence. By contrast, in the high-risk group, 2.5 times more patients (54.5%) had a recurrence of the disease and 50.0% of these tumours progressed.
Disease recurred in 9 (33.3%) patients with a low-risk chromosomal pattern after a mean observation time of 29.7 months. In the 18 (66.7%) patients with a high-risk pattern disease recurred within a mean 17.6 months. Although only 1 (11.1%) patient in the low-risk group progressed from a G1 to a G3 tumour within 36 months, 9 (50%) patients progressed in the high-risk group. In fact, the progression-free survival time in the high-risk group is half that of the low-risk group. These findings are in accordance with previous reports on chromosomal aberrations of chromosomes 7, 9 and 176,25,26 and underline the previously reported prognostic relevance of the loss of p16 expression. The data are also concordant with recent studies that underline the prognostic relevance of multicolour FISH for identifying patients at higher risk of progression, although on smaller patient cohorts and with a shorter observation time.27–29
Compared with previous studies,6,13,14 which reported a worse prognosis and decreased progression-free survival in patients with loss of p16 expression, our study shows that patients with a low-risk pattern have a longer disease-free survival time than patients in whom chromosomes 7 and 17 are affected. The differences may be caused by variabilities in tumour cohorts and different techniques as well as different study designs. On the other hand, our data also indirectly confirm the previously reported prognostic relevance of Ki-67 and p53,6,14,30,31 as their respective genes are located on chromosomes 7 and 17.3,32 Furthermore, they confirm that patients with a benign chromosomal pattern can be followed up at longer intervals following the EAU guidelines19 for patients at low risk of recurrence and progression.
CONCLUSION
With the use of the FISH technique, it is possible to assess the biological behaviour of urothelial carcinomas more accurately. Patients with a high-risk chromosomal pattern have a markedly shorter disease-free survival time and higher progression rate than those with a low-risk pattern. This was confirmed by a marked difference between the two groups in the Kaplan–Meier curve. Patients at higher risk of recurrence and progression could therefore be treated more aggressively to prevent tumour spread.
REFERENCES
Footnotes
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Competing interests: None.