Affiliations of authors: P.-W. Chiang (Department of Pediatrics), D. M. Kurnit (Departments of Pediatrics and Human Genetics), University of Michigan Medical Center, Ann Arbor; A. Schneider, S. Borgnat, M.-P. Gaub, P. Oudet, D. Jacqmin, Hopitaux Universítaires de Strasbourg Molecular Biology and Urology Departments, Strasbourg, France.
Correspondence to: David M. Kurnit, M.D., Ph.D., University of Michigan Medical School, MSRB I, Rm. 3520, Ann Arbor, MI 48109-0650 (e-mail: sesame{at}umich.edu).
Aberrations of the ratio between the copy numbers of two alleles of heterozygotes (microsatellite analyses) (15) or between the copy numbers of two different genes (quantitative polymerase chain reaction [QPCR]) (68) can be used to detect the abnormal genomes of cancer cells.
In this study, we found that these molecular methods were more sensitive than cystoscopy and cytology for monitoring the therapy of bladder tumors. This work was institutionally approved, and all subjects gave written informed consent.
Urine samples were obtained from all subjects, including controls, by spontaneous voiding or through the cystoscope. For cytology, urine samples were passed through Millipore filters (5-µm-pore size; Millipore Corp., Bedford, MA). We performed microsatellite analyses on urine sediment by using a set of 17 microsatellite probes as described previously (5). A positive microsatellite result was obtained when one or more allele ratios (of the 17 loci tested) fell outside the confidence interval as described previously (5). We also examined, by QPCR, ratios between copy numbers of three different genesp16/erbB-2, p16/SUPT4H, and erbB-2/SUPT4Has described previously (9); abnormalities were conservatively defined as a value of 2.0 or greater or less than or equal to 0.5 of the ratios. None of 32 control blood DNAs were found to be abnormal by QPCR analysis, demonstrating that this technique can be used to detect abnormal tumor DNA without substantial background from normal cells (data not shown).
We first studied 41 individuals with bladder cancer diagnosed by cystoscopy. By microsatellite analysis of 17 markers, urine sediments from 85% (35 of 41) of the samples from these cases were found to be abnormal. We did not observe a unique and simple pattern of imbalance for a given histologic subtype. Rather, different microsatellite loci were modified in different subjects with a given tumor type. The sensitivity for QPCR analysis based on the ratio between one probe pairing (erbB-2 and p16) was 44% (18 of 41). The six tumors that were undetected by both molecular methods were small; five of the six represented stage pTa disease, and the remaining sample was a pT1 tumor (10).
Multiple samples taken over time from 19 subjects were analyzed both by molecular (microsatellite analysis and QPCR) and by histologic (cystoscopy and cytology) criteria (Table 1). For QPCR, we achieved approximately 40% efficiency on patient samples known to be abnormal by microsatellite analysis using 17 markers (data not shown). In each of these 19 cases where the primary or recurrent tumor was available before therapy, both molecular results and cystoscopic results were abnormal initially. However, analysis of samples from follow-up visits presented a different picture.
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In addition, in two patients under treatment (i.e., bladder instillation of BCG or mitomycin C), the molecular markers remained positive one or two times with continued normality of cystoscopic results. For one of these patients, the abnormal molecular result was also concordant with abnormal cells seen on cytology, even though the cystoscopy gave normal results. Both patients later had normal molecular, cystoscopic, and cytologic results. This observation is consistent with the presence of residual disease evident at the molecular level that disappeared with time under treatment.
Both molecular and cystoscopic analyses gave normal results in three individuals. These samples were taken at later times during treatment and gave normal results by cystoscopy as well. This finding is consistent with successful extirpation of the bladder cancer by treatment.
For both microsatellite analysis and QPCR analysis, different modified loci were detected over time. There was no apparent association between the anomalies detected by microsatellite analysis or QPCR analysis. The molecular abnormality seen often varied at different time points in the same patient. In some cases, abnormalities were observed for at least some of the markers that had been abnormal earlier. In other cases, there was no obvious relationship, as was manifest in a previous study of bladder tumors (12). Thus, bladder cancer often results in desquamation of tumor cells over time. The molecular anomalies of these cells do not follow the rule of clonal generation of cancer cells documented elegantly in colon (13) and esophageal (14) malignancies. Therefore, diagnosis of whether or not a bladder tumor is present requires a thorough analysis of urine sediment at each time point.
As discussed above, molecular biologic and cystoscopic investigations usually show abnormal results before therapy is initiated. Initial cystoscopy as part of transurethral resection of the bladder will reveal the type, stage, and grade of the tumor (which can only be established histologically). However, once therapy is initiated, cystoscopy often shows normal results when the molecular biology is abnormal (16 of 32 cases, Table 1). The increased sensitivity of molecular methods compared with cystoscopy argues for the use of molecular techniques to monitor patients during therapy. We found that molecular and cystoscopic analyses of urine sediment diverged (Table 1
). As detailed in the text, many false-negatives based on cystoscopy occur, inasmuch as the molecular methodology gave positive results for 32 samples (serial samples from a number of individuals), while cystoscopy gave positive results only 16 times. Since other criteria showed that these patients often harbored bladder cancers, the cystoscopic results on these subjects were indeed falsely negative.
In summary, using molecular methods on sediment from readily obtained urine samples, we made the following observations: 1) The abnormal markers of genomic instability from desquamated bladder cancer cells changed over time; and 2) molecular methods (microsatellite analysis and QPCR analysis) were more sensitive than traditional methods (cystoscopy and cytology) in monitoring bladder tumors during therapy.
NOTES
Supported by La Ligue Nationale Contre le Cancer Section du Haut-Rhin (to P. Oudet and D. Jacqmin); by le groupe AZUR (P. Oudet and D. Jacqmin); and by Public Health Service grants R01CA78853 and R42CA77135 (to P.-W. Chiang and D. M. Kurnit) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
We thank Odile Regine for her constant and efficient technical performance of the microsatellite analysis.
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Manuscript received February 7, 2000; revised July 11, 2000; accepted August 14, 2000.
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