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Clinical Cancer Research Vol. 8, 35-40, January 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Microsatellite Analysis of Free Tumor DNA in Urine, Serum, and Plasma of Patients

A Minimally Invasive Method for the Detection of Bladder Cancer

Michael Utting1, Wolfram Werner, Regine Dahse, Jörg Schubert and Kerstin Junker

Department of Urology [M. U., W. W., J. S., K. J.] and Institute of Human Genetics and Anthropology [R. D.], Friedrich-Schiller-University Jena, 07743 Jena, Germany


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Purpose: Tumor cells may release DNA into circulation, which is subsequently carried as free DNA and enriched in blood and urine. The detection of tumors by microsatellite analysis of free DNA offers a possibility to establish a minimally invasive method for the detection of bladder cancer.

Experimental Design: We performed microsatellite analysis of free DNA of urine, serum, and plasma in comparison with DNA of lymphocytes and tumors of 40 patients with conspicuous bladder lesions. Six microsatellite markers were used for the detection of alterations on chromosomes 4, 9, and 17.

Results: Twenty-six of 36 bladder tumor tissue samples showed alterations. Microsatellite changes matching those in the tumor tissues were detected in at least one of the body fluids in 23 cases.

Conclusions: The study indicates that simultaneous and multiple investigations of microsatellite markers on free DNA of urine and blood could have clinical relevance as a minimally invasive method for diagnosis and screening of bladder cancer.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Presently, cystoscopy and direct biopsy are often performed to detect transitional cell carcinoma of the bladder (1) . However, the sensitivity of these methods is not satisfactory for detection of low-grade tumors and may yield up to 50% of false negatives (2) . Also, urine cytology is characterized by low sensitivity in the diagnosis of G1 tumors.

A successful treatment of bladder cancer depends on early detection and more specific approaches. It is widely accepted that human neoplasms arise as a result of the accumulation of multiple genetic alterations from mutations respectively. DNA replication errors (3) . Molecular genetic analyses of bladder tumor tissues have identified abnormalities in a number of chromosomes that appear to be involved in the development of tumors (4, 5, 6) .

Microsatellites are highly polymorphic DNA-repeat regions and common to all of the eucaryotic genomes. Alterations of microsatellite DNA are an integral part of neoplastic progression and are valuable as clonal markers for the detection of human cancers (6) . Using polymorphic microsatellite markers, it has been possible to detect LOH2 or DNA instabilities (microsatellite instability shift). A frequent LOH has been reported for bladder carcinoma in regions of chromosomes 4, 5, 8, 9, 11, and 17 and is considered a major event in the carcinogenesis of bladder cancer (7, 8, 9, 10, 11, 12) . LOH of chromosome 9 is the most frequent event described for bladder cancer (13, 14, 15) ; aberrations on chromosome 4 were reported for primary bladder carcinoma as well (9 , 16) , and alterations on the p-arm of chromosome 17, the locus of the p53 gene, have been described for invasive tumors (17) . In the present study, six microsatellite markers on chromosome 4, 9, and 17 were investigated.

Tumor cells may release DNA into the circulation so that increased quantities of DNA are found in the plasma of cancer patients compared with healthy controls (18 , 19) . The detection of microsatellite alterations of free DNA of body fluids seems a promising method and a sensitive tool for the detection of bladder cancer.

The purpose of this work was to evaluate a non-resp. minimally invasive procedure for the early detection of bladder carcinomas by MSA of free DNA from urine, serum, and plasma of patients.


   Materials and Methods
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 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Patients.
The samples of 40 patients with conspicuous bladder lesions (11 females, 29 males; average age, 69 years) were treated by transurethral resection between 1996 and 1999. Thirty-six of the patients tissue samples were defined as bladder tumors (20 x pTa, 8 x pT1, 8 x pT2–4, tumor classification according to the Union Internationale contre le Cancer, UICC), and used for additional investigations. In addition, 15 healthy controls with no history of cancer were recruited.

Sample Treatment.
Primary tumor tissues were obtained intraoperatively, frozen immediately after collection, and stored at -80°C. Frozen tissues were dissected at 5–10 µm, mounted on glass slides, and stained by H&E (20) . After examination by a pathologist, tumor cells were microdissected.

Blood and urine specimens were collected preoperatively by venipuncture and spontaneous micturition. The blood samples were centrifuged at 4,000 x g for 10 min, serum and plasma were carefully removed from plain and EDTA-containing (EDTA KE/9 ml; Monovette, Sarstedt, Germany) tubes, respectively, transferred into plain polypropylene tubes and stored at -80°C. Urine samples were fractionated and stored in 2.5-ml polypropylene tubes at -80°C. After defrosting, body fluid samples were centrifuged at 15,000 x g for 30 min. Only supernatants were used for analysis. Peripheral lymphocytes of each patient were used as a source of normal NA.

DNA Isolation.
DNA was isolated according to the protocol of QiaAmp Blood and Tissue kit (Qiagen, Hilden, Germany) and stored at 4°C.

Primers.
The following primer pairs were used for MSA: (a) FGA.PCR2 (nt2912); (b) MH34 (D4S243); (c) 401 (D9S747); (d) AFM186xc3 (D9S171); (e) nm00089 (D17S695); and (f) c17m135rp (D17S654; Ref. 21 ). The 5'end of one primer of each pair was labeled with the fluorescent dye IRD 800 (MWG Biotech, Ebersberg, Germany).

DNA Dilution Experiments.
DNA of two tumors and of lymphocytes of a control person were used for dilution series from 20 ng to 0.3 fg of template/polymerase chain reaction (dilution factor 2).

PCR.
PCR reactions were performed in 200-µl tubes at a total volume of 15 µl. The reactions were performed in Pre-mix Buffer F (100 mM Tris-HCL, 100 mM KCL, 400 µM each dNTP, 7.0 mM MgCL2, pH 8.3) (Biozym, Oldenburg, Germany) with 1.6 pmol of each primer, 0.5 units of Expand High Fidelity PCR System Taq-Polymerase (Roche, Mannheim, Germany), and 20 ng of template. Amplification protocol has an initial denaturation for 3 min at 94°C, 45 cycles for body fluids resp. 27 cycles for tumor tissues and lymphocytes; loop is 30 s at 94°C, 35 s at 58°C, and 30 s at 72°C, final elongation at 72°C for 5 min.

Denatured PCR products (1:1 in formamide/EDTA) were separated on 5% polyacrylamide gels (SEQUAGEL XR, Atlanta, GA) at 50°C and detected by laser fluorescence by LICOR machines (Lincoln, NE), equipped with RFLP scan software (Scanalytics, Billerica). A relative decrease of 70:30 of the signals of the alleles was scored as LOH.


   Results
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Genetic alterations were searched at six loci on chromosomes 4, 9, and 17 in samples of urine, serum, plasma, and tumor tissue in comparison with DNA of lymphocytes.

Alterations could be detected in 26 of the 36 patient bladder tumor tissues (72%). Tumor DNA of 10 cases showed no alterations for all six of the markers. However, 23 of the 26 tumors showing DNA alterations could be detected in at least one of the body fluids with at least one of the six primer pairs used (88%).

The results of MSA of body fluids show the presence of free DNA in 97% of the samples. Alterations could be detected in 50% of the samples, of which 27% were identical to the dissected tumor. No alterations were shown in 47% of the samples (Table 1)Citation .


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  		Table 1 Summary of the raw data after MSA

MSA of 6 markers on chromosome 4, 9, and 17 of free DNA from urine (column 1–6), serum (column 7–12), plasma (column 13–18), and of tumor DNA (column 19–24) of 36 bladder cancer patients. Alterations of the tumor tissue DNA are shown in grey boxes with black and white letters (column 19–24) and alterations in body fluids are shown in grey boxes with white letters (column 1–24). The last two rows represent a summary of all detected alterations. (L, LOH; S, Shift; N, normal DNA, column 25: tumor classification, 3d row: numbering of markers according to "Materials and Methods.")

 
In urine, 33 tumor-specific alterations of 122 general alterations (27%), in serum, 30 of 93 (32%), and in plasma 31 of 113 (28%) were detected (Fig. 1)Citation . On an average, 27% of the alterations in body fluids were identical to specific alterations of the dissected tumors, indicating that most of the alterations found in body fluids were different to those of dissected tumors (Fig. 2)Citation .



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  		Fig. 1. Overview of the results of MSA of 36 cases with histologically confirmed bladder cancer. Genetic alterations were detected in 26 of 36 tumor tissues. Twenty-three tumors with alterations could be detected in at least one of the body fluids with at least one of the primer pairs used. Of the body fluids, 50% showed genetic alterations, from which 27% were tumor specific.

 


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  		Fig. 2. MSA of free DNA on six markers of patient 577. Comparison of the results of urine, serum, and plasma samples with those of tumor and lymphocytes. Mainly tumor-specific alterations could be detected (black triangles) as well as alterations not specific to that of the dissected tumor (gray triangle) and normal DNA (not labeled). In one sample, no PCR product was detectable at marker D9S171 probably because of the loss of both alleles at this locus (X). U, urine; S, serum; P, plasma; T, tumor; L, lymphocyte.

 
The most frequent microsatellite alterations were detected on two markers of chromosome 9 (D9D747:80% resp. and D9S171: 92%) and 17 (D17S695: 64% resp. and D17S654: 77%); the least frequent microsatellite alterations were detected on chromosome 4 (nt2912: 11% resp. and D4S243: 19%; Table 2Citation ).


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  		Table 2 Alterations of the single markers

List of alterations detected on the single markers. DNA was isolated from 36 patients with bladder cancer (white and dark-grey columns, absolute number of the detected alterations; light grey columns, detected alterations of the tumors found in body fluids in percent; tum. spec., tumor specific).

 
In DNA-dilution experiments, PCR products were detectable when >= 5 pg of initial template/reaction were used. In some of the diluted lymphocyte DNA samples, PCR products could be detected when 0.08 pg of DNA was available.

Neither a relation between detected DNA alterations of the investigated loci and tumor staging/grading nor tumor progression could be found. A summary of the results is shown in Table 1Citation and Fig. 1Citation .

In urine, serum, and plasma of controls, there were no products detectable after PCR, but normal DNA was found in 9.5% of the samples (Table 3)Citation .


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  		Table 3 MSA of the control group

MSA of free DNA of 15 healthy controls. In urine, serum, and plasma, after PCR, no products were detectable, but normal DNA was found in 9.5% of the samples (N, normal DNA).

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In the present study, free DNA isolated from supernatants of urine, serum, and plasma in comparison with tumor and lymphocyte DNA was investigated. The detection of free DNA in 97% of the samples of body fluids (versus 9.5% in the controls) confirmed the studies of Leon et al. (18) and Shapiro et al. (19) , which show that DNA is enriched in blood and urine of cancer patients compared with healthy ontrols.

It was possible to detect at least one of the alterations of the 26 tumors in at least one of the corresponding body fluids with at least one of the six primer pairs used in 23 cases (88%; Table 1Citation ). However, tumor-specific alterations were detectable in 27% of free DNA samples, 73% of which showed alterations not found in the corresponding tumors (Table 1)Citation . The interpretation of PCR products obtained from circulating DNA requires special caution, especially when there is no DNA of tumor tissue available for analyses and comparison. The results of MSA depend on the quantity of free DNA as well as on its isolation protocol. Coulet et al. (22) have discussed PCR artifacts attributable to limited amounts of template DNA. However, in our hands, it was possible to detect PCR products even if >=5 pg of DNA were used. Moreover, PCR-products could be obtained in some of the samples when 0.08 pg of template was available.

The "real" status of circulating DNA in body fluids is not known, and the results of MSA of free DNA depend also on its isolation protocol. Free DNA is fragile and may be fragmented in body fluids leading to results difficult to interpret. It has been reported that free DNA found in plasma of pancreatic cancer patients shows variations in length of soluble DNA fragments between 88 bp and 80,000 bp (23) . Moreover, the DNA isolation method used for body fluids itself could represent a problem, because samples are centrifuged using DNA-binding columns, where shearing forces may fragment the DNA. For example, DNA may break close to or in the marker sequence, rendering interpretation of the results problematic. The concentration of free tumor DNA in body fluids is very low, so that even small losses of free tumor DNA because of fragmentation could represent a problem for a successful PCR amplification. In addition, free DNA may also be masked by proteins or other molecules circulating in the body fluids and, therefore, may not be available/accessible for MSA.

It should be emphasized that the isolation of free DNA of body fluids represents the basis for any additional investigation. The main focus of the known isolation methods is the isolation of the total amount of DNA regardless of its status. Therefore, new DNA-isolation techniques are necessary. Perhaps techniques involving a selective DNA "fishing" step without centrifugation may help to circumvent the above mentioned problems.

However, it is possible that the samples could be contaminated by normal DNA, which may mask the results of MSA (24) . To reduce these DNA contaminations, all samples of body fluids were centrifuged before DNA isolation, and only supernatants were used for analysis. This way, the amount of free DNA per sample could be increased in contrast with experiments where sediments were used in which mainly normal DNA was detectable (25) . However, normal DNA was still detectable in 47% of supernatants of body fluids and in 71% of the tumor tissue samples. Although a possible contamination of the body fluids with normal DNA cannot be completely excluded, the present tumor DNA does not seem to be altered at the markers investigated. At present, there are no enrichment techniques for an efficient isolation of free tumor DNA from body fluids available, and there is also no possibility to distinguish between the free normal DNA and the free tumor DNA.

Also, free DNA can originate from other sources such as different cells of multifocal and oliclonal tumors or from cells of a heterogeneous tumor. This might be a reason for the detection of alterations that are not found in the tissue of corresponding tumors.

However, alterations were detectable in only 29% of all of the tumor tissue samples. This indicates that the results of MSA depend largely on the selected markers. The markers should be highly specific in regard to the tumor and be stable in body fluids as well as after DNA isolation. To increase the probability for obtaining a specific result, MSA should be carried out on a high number of multiple markers of different chromosomes.

The present study shows that MSA of circulating DNA by parallel investigations of urine, serum, and plasma facilitates the detection of alterations in free DNA. However, the specificity of the markers is crucial for the diagnosis of bladder tumors (1) and individual prognosis (2) , i.e., the differentiation between low and high stage resp. grade tumors.

For a clinical usefulness of MSA of free DNA from urine, serum, and plasma as a minimally invasive method for the early detection and for an individual prognosis of bladder cancer, new DNA isolation techniques as well as multiple and parallel investigations of highly sensitive markers are necessary.


   ACKNOWLEDGMENTS
 
We thank Dr. Philippe Anker (University of Geneve, Geneva, Switzerland) and Dipl. Biol. Kathrin Reichwald (Institute of Molecular Biotechnology, Jena, Germany) for helpful discussions and proofreading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Institut of Molecular Biotechnology, Department of Genome Analysis, Beutenbergstrasse 11, D-07745 Jena, Germany. Phone: +493641-656482; Fax: +493641-65 62 55; E-mail: mutting{at}imb-jena.de Back

2 The abbreviations used are: LOH, loss of heterozygosity; MSA, microsatellite analysis; PCR, polymerase chain reaction. Back

Received 4/16/01; revised 10/22/01; accepted 10/30/01.


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 Materials and Methods
 Results
 Discussion
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