Affiliations of authors: J. Zhang, Z. Fan, Y. Gao, Q. An, S. Cheng (Department of Chemical Etiology and Carcinogenesis), Z. Xiao, C. Li (Department of Urology), Cancer Institute (Hospital), Chinese Academy of Medical Science and Peking Union Medical College, Beijing.
Correspondence to: Shujun Cheng, M.D., Department of Chemical Etiology and Carcinogenesis, Cancer Institute (Hospital), CAMS and PUMC, P.O. Box 2258, Beijing 100021, People's Republic of China (e-mail: chengsj{at}pubem.cicams.ac.cn
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One potential procedure that fulfills these requirements is microsatellite analysis. Alterations of microsatellites, the tandem repeat DNA sequences, are among the multiple genetic changes that take place during the development of primary bladder cancer (35). Mao et al. (6) and others (7,8) showed that microsatellite analysis of voided urine sediments has high sensitivity and specificity for the detection of primary and recurrent bladder cancers. However, because the number of patients with and without bladder cancer investigated in studies by Mao et al. (6) and by others (7,8) was small, the practical value of microsatellite analysis needs to be further assessed. Furthermore, the patients in these studies were all from the United States, and the potential of microsatellite analysis to detect bladder cancer in other ethnic groups is unknown.
In this study, we explored the practical value of microsatellite analysis for detecting bladder cancer in a Chinese population. We determined whether the microsatellites that are useful in detecting bladder cancer in the Chinese are similar to those that are useful in detecting bladder cancer in Western patients.
![]() |
PATIENTS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blood, urine, and/or tissue samples from 66 patients with bladder tumors were obtained from the Department of Urology, Cancer Institute (Hospital), Chinese Academy of Medical Science, Beijing. The tumors included transitional cell carcinomas (52 cases), adenocarcinomas (four cases), sarcomatoid carcinomas (two cases), squamous cell carcinoma (one case), inverted papillomas (five cases), neurofibroma (one case), and lipoma (one case). We also obtained blood and urine samples from six patients with non-neoplastic diseases of the bladder, including cystitis (three cases), calcification (one case), amyloidosis (one case), and granuloma (one case). In addition, we obtained blood and urine samples from one patient with lung cancer and from five healthy volunteers. The pathologic diagnosis was confirmed on all tissue samples. Surgical specimens were obtained by transurethral resection of bladder tumors, radical cystectomy, or nephroureterectomy. Peripheral blood and urine samples were collected before the patients underwent cystoscopic examination. All 78 patients and volunteers signed separate informed consent forms for sampling and DNA isolation and storage. The experiment procedures were reviewed and accepted by the ethics committees of the Cancer Institute (Hospital) of the Chinese Academy of Medical Science. Sex, age, and clinical data for all patients are summarized in Tables 1 and 2.
|
|
To obtain leukocytes, 5 mL of peripheral blood was treated with citrate and the final concentration of citrate was 0.2%. The blood was then diluted with lymphocyte-isolating solution (Institute of Haematology, Chinese Academy of Medical Science, Tianjin, China) and was centrifuged at room temperature at 8001000g for 10 minutes. The layer containing leukocytes was collected and washed twice with phosphate-buffered saline (PBS). Cell pellets were stored at -80 °C until DNA extraction.
To obtain urine sediments, about 50200 mL of urine was centrifuged at 4 °C at 4000g for at least 15 minutes. The sediment pellet was washed twice with PBS and stored at -80 °C until DNA extraction.
In each case, fresh tumor samples after dissection were frozen immediately in liquid nitrogen for 5 minutes and stored at -80 °C until DNA extraction. Ten formalin-fixed, paraffin-embedded tumors from nine patients were processed as described previously (9).
DNA Preparation
Frozen tumor tissue was pulverized in the presence of liquid nitrogen. Pulverized tumors, leukocytes, and urine-sediment pellets were digested with 50 µg/mL proteinase K in 1% (wt/vol) sodium dodecyl sulfate for 814 hours at 48 °C and extracted twice with phenol/chloroform. The DNA was then precipitated with two volumes of 95% ethanol and 1/3 volume of 3 M sodium acetate. Ten formalin-fixed, paraffin-embedded histologic sections from nine patients were microdissected to enrich for tumor cells, and the DNA was isolated as described previously (9).
Microsatellite Analysis
DNA from each sample was analyzed by use of 60 microsatellite markers (Research Genetics, Huntsville, AL), including 13 markers described by Mao et al. (6) and by others (7,8), by use of the polymerase chain reaction. Briefly, the forward primer of each marker pair was end labeled with [-32P]deoxyadenosine triphosphate (Du Pont NEN, Boston, MA). In each polymerase chain reaction, 20 ng of DNA was subjected to 35 cycles, in which each cycle consisted of 95 °C for 30 seconds (denaturating temperature), 52 °C62 °C for 1 minute (varying annealing temperature), and 72 °C for 1 minute (extension temperature). There was a single final extension at 72 °C for 10 minutes. Polymerase chain reaction products were separated by electrophoresis on a denaturing 7% ureapolyacrylamideforamide gel, and the bands were detected by autoradiography.
Informative cases, defined as those in which the length of two alleles is different and can be separated by polyacrylamide gel electrophoresis, were scored as a loss of heterozygosity (LOH) if we detected a reduction of at least 50% in one allele compared with the normal (germline) allele (Fig. 1). The presence of a new or abnormal allele in the tumor or urine DNA was scored as a "shift," as shown in Fig. 1
. Initially, LOH was screened visually for loss of an allele or the presence of microsatellite shifts, and cases with a "partial loss" were evaluated further by use of the public domain of the National Institutes of Health (NIH) (Bethesda, MD) Imagine Program (developed at the NIH and available on the Internet at http://rsb.info.nih.gov/nih-image). All results were confirmed by at least two independent observers.
|
Using the 60 microsatellite markers, we first analyzed DNA from leukocytes (normal control), tumor specimens, and urine sediments from 36 patients with bladder cancer and from two with inverted papilloma (Table 1, cases 138). Nine markers giving clearly readable patterns and susceptible to alterations were combined in a panel that could detect the greatest number of primary tumors.
To determine the specificity and the sensitivity of the panel of microsatellite markers and to investigate its diagnostic value, we performed microsatellite analyses by using the nine selected markers on urine-sediment DNA samples from 40 individuals with or without suspicious bladder lesions (Table 2, cases 3978). Laboratory investigators were blinded to clinical information, and the final pathologic diagnosis and laboratory results were not disclosed to clinical investigators until all of the data had been collected and analyzed.
Statistical Analysis
Chi-square tests were used to evaluate the relationship between alterations of a given marker and tumor stage or grade. Chi-square tests and the calculations for specificity and sensitivity were performed by use of the software package Epi Info version 5.0 (Epidemiology Program Office, Centers for Disease Control and Prevention, Atlanta, GA). All statistical tests were two-sided.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To set up a simple and practical panel for the detection of bladder cancer in the Chinese, we selected nine markers giving clearly distinct patterns (e.g., Fig. 1) and susceptible to alterations, which can detect the same number of primary tumors as using all of the 60 markers (Tables 1 and 2
).
Nontumor cell contamination from stromal or inflammatory cells is common in tumor masses and can influence the results of genetic analyses, such as the LOH or microsatellite analyses. This problem existed in some of the tumor masses examined in this study because we did not identify any microsatellite alterations at any of the 60 selected microsatellite markers in the frozen tissue of nine of the 38 tumors (marked with a in Table 1
). To exclude the possibility that the tumor DNA was being "diluted" by contamination with DNA from nontumor tissue, we microdissected the paraffin-embedded histologic sections of these tumors and reanalyzed the tumor DNA. Among the nine tumors, three (Table 1
, cases 2, 4, and 38) had microsatellite alterations at several loci, indicating that non-neoplastic contamination had led to false-negative results. However, no microsatellite alterations were detected in either the frozen or the microdissected tissue from the other six tumors.
To determine the specificity and the sensitivity of the panel of microsatellite markers and to investigate the practical value of the panel in detecting bladder cancer in Chinese patients, we performed the microsatellite analyses in a blinded fashion on urine-sediment DNAs from 40 individuals with and without various bladder pathologic abnormalities, including bladder cancer. We did not learn the status of each patient until the analysis was complete. Microsatellite alterations were detected in the DNA from 22 (96%) of 23 patients with bladder cancer and from all three patients with inverted papilloma but not in the DNA from the patient with lipoma or from the patient with neurofibroma. Furthermore, microsatellite alterations were not detected in any of the five healthy volunteers, in the six individuals with non-neoplastic diseases of the bladder, or in the patient with lung cancer (Table 2). The results demonstrated that the microsatellite analysis of voided urine with the nine-marker panel is a practical method for detection of bladder cancer in the Chinese, with excellent sensitivity (96%) and specificity (100%).
A major concern for routine urine cytology is its poor sensitivity, particularly for low-grade tumors. To address this concern, we compared the sensitivity of microsatellite analysis on urine-sediment DNA with routine urine cytology. Microsatellite analysis of urine sediment DNA and routine urine cytology were performed in a blinded fashion on 17 patients with bladder cancer (Table 2). Sixteen cases (sensitivity = 94%) of bladder cancer were detected by microsatellite analysis, while only eight cases (sensitivity = 47%) were detected by cytology (Table 2
). The data demonstrate that microsatellite analysis of urine-sediment DNA is more sensitive than a routine urine-cytology examination. We did, however, detect bladder cancer in one patient (case 47) by cytology but not by microsatellite analysis (Table 2
). Similar results were also observed previously by Mao et al. (6). When we combine microsatellite analysis on urine-sediment DNA with routine urine cytology, we can detect all of the 17 patients with bladder cancer.
Initially, we analyzed 38 bladder tumors and corresponding urine sediments by use of 60 microsatellite markers and selected nine markers, most of which were different from those markers used for the U.S. population, for further analysis. In the subsequent blinded study, microsatellite analysis with this nine-marker panel was highly sensitive (22 [96%] of 23) and specific (100%) for the detection of bladder cancer in the Chinese. Most of the microsatellite alterations detected in urine-sediment DNA agreed with those from tumor DNA, indicating that the microsatellite abnormalities in urine sediments could represent the abnormalities of tumor DNA. Among the alterations detected, LOH was more common than a shift for any given marker, which is in accordance with previous reports (68). We found no statistically significant associations between alterations of any microsatellite markers and sex, age, and tumor type and stage or grade (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The microsatellite marker panel used in this study consisted of nine microsatellites distributed on five chromosomes, specifically, chromosomes 5, 9, 16, 17, and 18. LOHs of these chromosomes are known frequent events during tumorigenesis. Changes at any given microsatellite marker were not associated with tumor stage or grade (data not shown). Although one reason for the lack of association between tumor grade or stage and microsatellite alterations may be due to the small number of samples, our results suggest that the panel of microsatellite markers might also be useful in the detection of early-stage bladder cancer in Chinese patients.
One of the initial goals of this study was to determine if microsatellite analysis is an effective screening tool for the detection of bladder cancer. A comparison of microsatellite analysis of urine sediments with routine cytology shows several advantages to the former. In particular, microsatellite analysis proved to be highly specific and highly sensitive. However, the results of microsatellite analysis, a technique based on abnormalities of tumor DNA, may be affected either by contamination with nontumor DNA (dilution effect) or by the limitation that the microsatellites are located in regions of DNA that do not contain any abnormalities. For example, case 38 is a patient diagnosed with a pT3b-stage transitional cell carcinoma with severe hematuria. Several microsatellite markers had alterations in the tumor sample but not in the urine-sediment sample. The difference in results may reflect a high ratio of lymphocyte DNA to tumor cell DNA that subsequently diluted the detection of tumor-genetic abnormalities in the urine. Another example is case 35, a patient with pT3b-stage squamous cell carcinoma who had no alterations in either tumor or urine-sediment samples. Although the panel of microsatellites examined had no alterations, the possibility that other microsatellites not tested had alterations cannot be ruled out. This result would, therefore, reflect a limitation of the markers selected. Cytologic examination does have one advantage over microsatellite analysis: It is a morphologic method that can provide a clue to doctors even if only a single cancer cell is detected. This may explain why case 38 could be detected by cytology despite heavy lymphocyte contamination. Of course, for most bladder cancer patients, tumor cells in urine are abundant and the number easily meets the need for successful microsatellite analysis.
It is of interest that the microsatellite panel selected in this study is different from that reported by Mao et al. (6) and by others (7,8). Although we initially screened the same panel as used by Mao et al. (6), the high sensitivity and specificity achieved with our subsequent panel suggest that apparent ethnic and potential etiologic differences between Chinese and U.S. patients are reflected in microsatellites. In addition to microsatellites D9S171 and FGA (Fig. 2), we also identified several other microsatellite markers, including D18S984, D17S855, and D3S1235, that showed a much lower heterozygous rate in Chinese (Fig. 2
) than in Western patients (Genome Database http://www.gdb.org and Research Genetics Company MapPairs website http://www.resgen.com/resources/apps/mappairs/index.php3). This result demonstrated ethnic differences at a genetic level between the Chinese and the U.S. populations. In addition, although we found that D9S747 showed a similar heterozygous rate in Chinese patients similar to that in Western patients (68), this marker had fewer alterations in Chinese patients (data not shown). This observation possibly reflects etiologic differences between Chinese and U.S. patients, e.g., differences in the environmental carcinogen exposure, differences in dietary habits, and differences in social behavior. Although some markers were not useful for the detection of bladder cancer in Chinese patients, other markers, such as D17S695, D16S476, and D9S162, were useful for the detection of bladder cancer in both Chinese and U.S. patients (68). These markers may reflect common genetic changes that occur during the development of human bladder cancer.
Tumorigenesis is a multistep process with multiple genetic changes (10,11). The pattern of genetic abnormalities of a given tumor may be unique among different ethnic groups because of the differences in genetic background, etiology, and/or molecular pathology. We have identified a distinct microsatellite panel that is sensitive and specific for the detection of bladder cancer in Chinese patients. Conceivably, differences may exist for genetic markers other than microsatellites and, therefore, must be considered when selecting markers for genetic analysis in different ethnic populations.
![]() |
NOTES |
---|
We thank Dr. Jin Jen of The John Hopkins Oncology Center, Baltimore, MD, Dr. Nan Hu of the National Institutes of Health, Bethesda, MD, and Dr. Li Mao of the M. D. Anderson Cancer Center, Houston, TX, for their stimulating suggestions and valuable technical assistance.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1
Wingo PA, Tong T, Bolden S. Cancer statistics, 1995. CA Cancer J Clin 1995;45:830.
2 Koss LG, Deitch D, Ramanathan R, Sherman AB. Diagnostic value of cytology of voided urine. Acta Cytol 1985;29:8106.[Medline]
3 Knowles MA. Molecular genetics of bladder cancer: pathways of development and progression. Cancer Surv 1998;31:4976.
4 Knowles MA, Elder PA, Williamson M, Cairns JP, Shaw ME, Law MG. Allelotype of human bladder cancer. Cancer Res 1994;54:5318.[Abstract]
5 Gonzalez-Zulueta M, Ruppert JM, Tokino K, Tsai YC, Spruck CH 3d, Miyao N, et al. Microsatellite instability in bladder cancer. Cancer Res 1993;53:56203.[Abstract]
6 Mao L, Schoenberg MP, Scicchitano M, Erozan YS, Merlo A, Schwab D, et al. Molecular detection of primary bladder cancer by microsatellite analysis. Science 1996;271:65962.[Abstract]
7 Steiner G, Schoenberg MP, Linn JF, Mao L, Sidransky D. Detection of bladder cancer recurrence by microsatellite analysis of urine. Nat Med 1997;3:6214.[Medline]
8 Linn JF, Lango M, Halachmi S, Schoenberg MP, Sidransky D. Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients. Int J Cancer 1997;74:6259.[Medline]
9
Jen J, Kim H, Piantadosi S, Liu ZF, Levitt RC, Sistonen P, et al. Allelic loss of chromosome 18q and prognosis in colorectal cancer. N Engl J Med 1994;331:21321.
10 Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:7596.[Medline]
11
Kohno T, Yokota J. How many tumor suppressor genes are involved in human lung carcinogenesis? Carcinogenesis 1999;20:140310.
12 Mostofi FK. Histological typing of urinary bladder tumors. In: Mostofi FK, Davis CJ, Sesterhenn IA, editors. In collaboration with L. H. Sobin and pathologists in 10 countries. 2nd ed. Berlin (Germany): Springer; 1999.
Manuscript received May 23, 2000; revised October 26, 2000; accepted October 30, 2000.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |