Microsatellite Alterations in Human Bladder Cancer:: Detection of Tumor Cells in Urine Sediment and Tumor Tissue
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.
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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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Prognostic value of loss of heterozygosity at chromosome 9p in nonmuscle-invasive bladder cancer
2010, UrologyCitation Excerpt :Observation of LOH at a specific chromosomal marker in cells from the tumor suggests the presence of a closely linked tumor suppressor gene, the loss of which is involved in pathogenesis of the tumor. In bladder cancer, LOH has been shown to involve several loci throughout the genome with chromosome 9 being the most frequently altered in bladder cancer.3-11 The ability to identify clonal population of tumor-derived cells in urine sediment has been demonstrated.
Quantitative loss of heterozygosity analysis for urothelial carcinoma detection and prognosis
2010, UrologyCitation Excerpt :In contrast, thresholds determined in the present study ensured negative LOH in all healthy controls and thus 100% specificity in UC patients. We observed LOH frequencies on individual markers that agree with the average 25% alteration rate per microsatellite locus reported by Berger et al10 as a reasonable threshold for clinical practice. Chromosome 9 was the most frequently involved, as previously reported6,11 as it is associated with the earliest stages of urothelial oncogenesis.12
Prognostic Role of Loss of Heterozygosity on Chromosome 18 in Patients With Low-Risk Nonmuscle-Invasive Bladder Cancer: Results from a Prospective Study
2010, Journal of Surgical ResearchCitation Excerpt :The data obtained from our study can be explained in consideration of the fact that LOH on Chr 18q22 is a late event in bladder tumorigenesis [24]. Finally, as suggested by Berger et al., LOH on Chr 18 should be considered a disease marker that could improve the diagnostic efficacy of current standard methods [25]. We found that only two out of 43 controls showed LOH on Chr 18, whereas a total of 38 out of the 65 affected patients showed LOH on Chr 18, with a significant statistical difference.
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2008, Journal of UrologyCitation Excerpt :Determination of the panel may have clinical relevance because most renal tumors are clinically assumed to be RCC and are treated using radical nephrectomy without an intraoperative diagnosis. Our study also differed from prior reports of microsatellite urinalysis13–20 because we comprehensively analyzed LOH in primary tumors and in their urine sediments using a total of 77 UC related markers from 25 chromosomal regions and 74 RCC related markers from 23 chromosomal regions. A large cohort study is mandatory to validate the diagnostic panel in patients with renal tumors before recommendation for clinical application.
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