Elsevier

European Urology

Volume 41, Issue 5, May 2002, Pages 532-539
European Urology

Microsatellite Alterations in Human Bladder Cancer:: Detection of Tumor Cells in Urine Sediment and Tumor Tissue

https://doi.org/10.1016/S0302-2838(02)00073-8Get rights and content

Abstract

Objectives: Bladder cancer is the result of clonal expansion of cancer cells in which multiple genetic alterations have occurred. Loss of heterozygosity (LOH) studies have demonstrated that alterations in microsatellite regions are common in bladder cancer. This observation offers the possibility of early tumor detection by examining the DNA of urinary sediment.

Methods: We investigated alterations of 17 microsatellite loci in urinary bladder carcinomas of different stages and grades. Per locus, 19–30 specimens were evaluated. DNA was isolated from tumor specimens, urinary sediment and peripheral blood lymphocytes. DNA fragments of 17 microsatellite loci were amplified by PCR and analyzed for genomic alterations.

Results: Microsatellite alterations were detected in tumor tissue and urine sediment from 27 out of 31 patients (87%). Urine sediment analysis alone proved positive in 24 out of 31 patients (77%). The type of lesions most frequently detected was LOH (74% of all alterations), followed by length alteration (24%) and additional alleles (2%). On average, the alteration frequency was 22% per locus. The loci at chromosomes 9 and 18 proved most informative. No alterations were found in grade I tumors. The study revealed a correlation between microsatellite alterations and the respective grades and stages of the tumors. Average alteration frequencies per locus were: 27.4% in grade III versus 19.3% in grade II tumors, 26.5% in invasive versus 12.3% in superficial tumors.

Conclusions: Our results demonstrate that microsatellite alterations are common in bladder cancer and that analysis of genomic instabilities in urine samples should be further evaluated as a method for bladder cancer screening in a high-risk group. Especially, when a set of microsatellites is used that shows a high probability of detecting alterations and allows easy handling, this could be an alternative or a completion to currently available methods.

Introduction

Initial diagnosis of bladder cancer and follow-up surveillance is based on cystoscopy, urine cytology, histological examination of bladder biopsies and imaging studies. However, false positive and false negative results are quite frequent because tumors have to attain a certain size to be detected by cystoscopy, urogram or biopsy, and the sensitivity of cytology depends on the tumor grade [1]. A sensitive and reliable tool for detection of bladder cancer is clearly warranted to improve early diagnosis. Several new diagnostic methods for non-invasive detection of bladder cancer are currently under investigation, some—the BTAstat test®, the BTAtrak test®, the NMP22® or the ImmunoCyt® assays—have recently been approved by the FDA [2].

Many tumors, among them carcinoma of the bladder, are characterized by increased chromosomal instability resulting in chromosomal abnormalities and loss of heterozygosity (LOH) [3]. These genetic losses can be detected by analysis of highly polymorphic markers, such as microsatellites [4], [5]. The elements of repetitive DNA sequences, containing for example (CA)n repeats, are scattered throughout the human genome [6]. Microsatellite analysis revealed genetic instability in many different tumors, such as lung cancer [7], squamous cell carcinoma or colorectal carcinoma [8].

Frequent occurrence of loss and length alteration of microsatellites has also been described for transitional cell carcinomas of the bladder. Several studies reported alterations on chromosome 9 as frequent and occurring early in both superficial and muscle-invasive bladder tumors [9], [10], [11], [12], [13], [14]. In addition, LOH is also found frequently on chromosomes 8 and 14. Various groups try to localize putative tumor suppressor genes that might be involved in bladder cancer carcinogenesis on these chromosomes [15], [16], [17], [18].

Analysis of alterations of microsatellite markers may provide a reliable, non-invasive method to detect malignant cells in the urinary sediment and that can be utilized for both follow-up of bladder cancer patients and screening of high-risk populations. In an attempt to create a set of microsatellite markers suitable for screening, we investigated markers described as particularly susceptible for instability in bladder cancer for their sensitivity in detecting tumor cells in urinary sediment.

In our study, we screened 17 microsatellite markers distributed on seven chromosomes in the DNA of 31 primary bladder carcinomas and in the sediment of the matching urine specimens. We looked for LOH, length alteration and additional alleles in these loci.

Section snippets

Materials and methods

DNA obtained from tumor specimens and urine sediments of 31 patients with transitional cell carcinoma was evaluated for microsatellite instability using a set of 17 microsatellite markers (13 dinucleotid-repeats and four tetranucleotid-repeats). The investigation focused on chromosomes 8, 9 and 14. In addition, markers from chromosomes 4, 11, 18 and 21 were included (Table 1). Tumor specimens investigated were 3 grade I, 12 grade II and 16 grade III malignancies; nine of the tumors were

Results

Altogether we carried out 405 comparisons between normal DNA (peripheral blood mononuclear cells) and the corresponding tumor DNA (urine sediment or tumor tissue) (Fig. 2).

Out of 405 normal-DNA versus tumor-DNA comparisons, we detected 91 microsatellite alterations (22.5%). These alterations can be divided into three subsets of microsatellite instabilities.

The most frequent type of lesion is LOH that occurred 67 times (73.6% of all alterations). With 22 length alterations (24.2% of all

Discussion

Multiple chromosomal deletions have been reported in several types of tumors, including bladder cancer. The present study confirms the observation of a high rate of microsatellite marker alteration occurring in transitional cell carcinoma of the bladder [17], [18], [22], [23]. Following the groundbreaking study of Mao et al. [24], who identified microsatellite changes in 95% in the urine sediments of bladder cancer patients, we also detected high rates of instabilities in tumor tissue and also

Conclusions

Recurrence of bladder cancer in a risk population can be detected by microsatellite marker analysis with little burden for the patient. From a practical point of view, it is necessary to perform the microsatellite analysis rapidly and reliably. Our results clearly indicate that certain microsatellite markers show a special susceptibility for instability. If these results are confirmed in larger studies, the use of a set of markers that show high rates of microsatellite alterations might be

Acknowledgements

The authors are grateful to Maria Pöschl for her technical assistance. This work was supported by the Jubiläumsfond of the Austrian National bank, Project 6706.

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