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Technical Briefs |
Departments of1 Clinical Chemistry,2 Epidemiology and Biostatistics, and3 Urology, University Medical Center Nijmegen, Nijmegen, The Netherlands;4 Department of Urology, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands
aaddress correspondence to this author at: AKC/564, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands; fax 31-243541743, e-mail i.schultz{at}akc.umcn.nl
Urothelial cell carcinoma (UCC) is the most common form of bladder cancer (1). Approximately 70% of patients diagnosed with UCC have superficial tumors (designated Ta or T1) and are treated by transurethral resection of the tumor (TURT). Most of these patients develop recurrences after TURT and therefore need to be followed intensively. Cystoscopy is the gold standard to check a patient’s bladder for recurrences. Unfortunately, cystoscopy is invasive, labor-intensive, and costly. Accurate prediction of UCC recurrence could significantly reduce the number of cystoscopies performed during patient follow-up.
Currently, the major determinants of a patient’s follow-up scheme and treatment procedures are pathologic tumor stage and grade. However, pathology-based assessment of bladder tumor stage and grade is subject to variability (2)(3). Furthermore, it is important to address other disease characteristics, such as tumor multiplicity and tumor size, to carefully assess the risk of recurrence in patients with superficial UCC (4)(5)(6). As a possibly better and more standardized estimation of the risk of recurrence in patients with UCC, molecular biological alterations have been studied (7)(8)(9)(10). An important molecular tumor marker that has emerged is survivin (11). The concentrations of both survivin protein and mRNA in tumor tissue from patients with superficial UCC are indicative of the risk of tumor recurrence (12)(13).
Assessment of prognosis in UCC is usually restricted to analysis of the resected tumor tissue. However, routine cystoscopy frequently overlooks carcinoma in situ and small, solid high-grade or papillary tumors (14)(15). These lesions may thus not be removed during TURT, and consequently, prognostic information may be missed. As an advantage over tumor biopsies, bladder washings cover the entire urothelium and may reflect the general molecular biological status of the bladder (16)(17)(18)(19). They are easily obtained during cystoscopy or before TURT.
We quantified survivin mRNA in bladder washings by real-time quantitative reverse transcription-PCR. The data were correlated with histopathologic characteristics and with time to first recurrence in patients with superficial UCC.
We collected thirty-seven 50-mL bladder washings [performed with phosphate-buffered saline (PBS)] before TURT from 36 patients with UCC. The patients were asked to participate in the study and gave oral informed consent. After collection, the washings were immediately cooled on ice, and the cells were harvested by centrifugation at 4 °C for 10 min at 800g. The cell pellet was washed twice with ice-cold PBS and subsequently resuspended in ice-cold PBS containing 15 mL/L fetal calf serum. We enriched the bladder washings for urothelial cells to eliminate possible contamination with (white) blood cells, which could interfere with normalization of gene expression, by adding 30 µL of magnetic beads (3 x 107 particles) coated with an antibody (Ber-EP4) against epithelial cells (Dynal). After incubation at 4 °C for 30 min, the bead-bound urothelial cells were washed three times with PBS containing 15 mL/L fetal calf serum, frozen in liquid nitrogen, and stored at –80 °C.
The histopathologic characteristics of the resected tumor tissues were evaluated by a pathologist according to the WHO criteria for tumor grade (20) and the TNM classification for stage (21). The tumors of two patients were classified as superficial and low grade, but the pathology report did not specify stage (Ta, T1) or grade (grade I, grade II). Patients were followed clinically from the time of tumor resection. The mean follow-up period was 32.6 months (range, 1–45 months). Thirteen of the 27 patients with superficial (Ta/T1) UCC received adjuvant treatment after TURT. The time to first recurrence was determined only for patients with superficial UCC.
Isolation of total RNA from the bladder washing cells and cDNA synthesis were performed as described previously (22). Subsequent real-time PCR quantification of survivin mRNA and, for normalization of expression, of the housekeeping gene cyclophilin A was performed as described previously in detail (13). The primers and the probe for survivin were chosen such that all three known splice variants (23) were detected. Real-time quantitative PCR experiments were performed with an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). We quantified survivin and cyclophilin A mRNA copy numbers by constructing linearized plasmids containing either of the amplicons. A triplicate 5-log-range calibration curve containing 10 to 106 copies of either survivin or cyclophilin was included in each real-time PCR assay.
Statistical analyses were performed with the Statistical Package for Social Sciences (SPSS), release 10.0.7 (June 2000). Only bladder washings with a cyclophilin A copy number 
5 were included in the analyses because lower numbers indicated insufficient yield of cells from the bladder washings and thus unreliable quantification of survivin mRNA copy number. Four of the 37 bladder washings did not meet this criterion and were excluded from further analyses.
We first investigated the correlation between normalized survivin mRNA copy number and tumor pathology characteristics, using the Kruskal–Wallis test (Fig. 1A
). The two patients for whom tumor stage and grade were not specified were excluded from this analysis. Although the Kruskal–Wallis P values were 0.001 and 0.004 for tumor stage and grade, respectively, indicating significant differences in expression (Fig. 1A
), we observed no linear correlation between survivin mRNA copy number and increasing stage or grade.
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We subsequently investigated whether normalized survivin mRNA copy numbers in bladder washings of patients with superficial UCC could be used to distinguish patients with a high risk of recurrence from patients with a low risk. One patient with superficial UCC died 1 month after TURT and was excluded from the analysis because the first control cystoscopy for detection of tumor recurrence is not performed earlier than 3 months after TURT. The two patients for whom the tumor stage and grade were not specified were included in this analysis because their medical files clearly stated that they suffered from superficial UCC. The median normalized survivin mRNA copy number for patients with superficial UCCs was 0.13, which was used as the cutoff value. Kaplan–Meier curves were constructed for patients with a normalized survivin mRNA copy number above (n = 13) and below (n = 13) the cutoff (Fig. 1B
). The difference between the curves was statistically significant (log-rank test, P = 0.018). The median times to recurrence for patients with a normalized survivin mRNA copy number above and below the median were 10 and 22 months, respectively.
In the low- and high-risk groups, 3 and 10 patients, respectively, received adjuvant treatment after TURT. Of the patients with superficial UCC and a normalized survivin mRNA copy number above the cutoff (n = 13), 3 patients died recurrence-free (after 3, 11, and 14 months). Eight patients had recurrences within 1 year, and one had a recurrence after 14 months. Another patient remained recurrence-free during the follow-up. Of the patients with superficial UCC and a mRNA copy number below the cutoff (n = 13), 1 patient died after 11 months (recurrence-free). Of the remaining 12 patients, only 1 had a recurrence within 12 months (after 10 months).
The results show that bladder washings provide good-quality patient material: 33 (89%) of our bladder washings yielded sufficient amounts of cells for reliable quantification of mRNA. The poor quality of four bladder washings may have been attributable to insufficient rinsing of the bladder and, consequently, a low yield of cells.
In addition, we investigated the possible relationship between survivin mRNA copy number in bladder washings and tumor pathologic grade and stage. Although previous studies showed that mRNA or protein concentrations of other molecular markers found in bladder washings can indicate UCC stage or grade (22)(24), we observed substantial overlap in the survivin mRNA copy numbers between the different tumor stages (e.g., between Ta and T2) and grades (e.g., between grade I and grade II). Therefore, in our study, survivin mRNA copy numbers in bladder washings did not seem directly indicative of UCC stage or grade.
We also investigated whether survivin mRNA could be detected in the urine from our patient group because collection of urine would circumvent cystoscopy. Despite immediate processing, mRNA could not be detected in 50% of the urines, and in cases in which mRNA was quantified, we found correlations with neither pathology nor clinical characteristics (data not shown).
Thus, bladder washings provide good-quality patient material, and quantification of survivin mRNA therein may indicate whether a patient needs early or late cystoscopy after TURT. If this can be confirmed in a larger cohort of patients, it may help to reduce the number of cystoscopies in patients with superficial UCC.
References
Ex3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. Cancer Res 1999;59:6097-6102.The following articles in journals at HighWire Press have cited this article:
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  P. C. Black, G. A. Brown, and C. P. Dinney Molecular Markers of Urothelial Cancer and Their Use in the Monitoring of Superficial Urothelial Cancer J. Clin. Oncol., December 10, 2006; 24(35): 5528 - 5535. [Abstract] [Full Text] [PDF]
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 I. J. Schultz, L. A. Kiemeney, J. L. Willems, D. W. Swinkels, J. A. Witjes, and J. B. de Kok Survivin and MKI67 mRNA Expression in Bladder Washings of Patients with Superficial Urothelial Cell Carcinoma Correlate with Tumor Stage and Grade but Do Not Predict Tumor Recurrence. Clin. Chem., July 1, 2006; 52(7): 1440 - 1442. [Full Text] [PDF]
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