Medical sciences Medicine Medizin MOLECULAR SEROLOGICAL DIAGNOSIS AND URINANALYSIS IN TRANSITIONAL CELL BLADDER CANCER Introduction and Objectives: For transitional cell carcinoma (TCC) of the urinary bladder no reliable serological markers and no sufficient non-invasive tools for urine diagnosis are available. We applied the fluorescent microsatellite analysis (MSA) to detect serum-DNA and urine-sediment-DNA alterations in patients suffering from bladder cancer. Materials and Methods: From 2001 to 2003 we prospectively collected fresh tumor-, peripheral blood-, serum- and spontaneous urine specimens from 117 consecutive patients treated for TCC of the bladder at our institution. DNA was extracted by phenol-chloroform method from tumors and blood lymphocytes. Free Serum- and cellular urine-sediment-DNA was isolated by a commercial kit (Mini-Kit, Qiagen). We performed MSA with a total of only 10 polymorphic markers from the chromosomal regions 5q, 8p, 9p, 9q, 13q, 14q, 17p, and 20q to identify tumor specific serum- and urine-sediment-DNA alterations. After PCR- amplification detection of allelic imbalance and loss of heterozygosity was carried out on an automated laser sequencer (ALFexpress II, Amersham- Pharmacia Biotech). 20 healthy controls were investigated with the same markers. Results: We identified serum-DNA alterations in 77.7% (87/112) of cases. By applying the same 10 microsatellite markers we observed tumor specific urine- DNA alterations allowing cancer diagnosis in 85% (64/75) of cases. Four healthy controls displayed serum-DNA artefacts rendering a specificity of 80%. The highest frequency of serum-DNA alterations was detected for chromosomal region 9p with 35%. The other Chromosomes showed serum-DNA alterations in 16 to 26%. In urine 9q displayed alterations most frequently in 37% of cases. The identification of serum-DNA alterations was associated with underlying Abstract 93 tumor–stage (p = 0.008) and was also more frequent in high grade tumors (p = 0.005). This was not the case for the urine-diagnosis (p ] 0.05). Conclusions: In patients with TCC of the urinary bladder microsatellite analysis with only 10 markers has a high sensitivity of 77.7% in the detection of free serum-DNA alterations, thus allowing tumor diagnosis. For urine-sediment specimens the detection rate is even 85% independent of tumor stage or grade. https://doi.org/10.17192/z2014.0264 ppn:337895589 2014-03-13 2014-03-18 2014 application/pdf monograph Philipps-Universität Marburg bladder alteration opus:5433 Urothelkarzinom, Mikrosatellitenalterationen Medizin Götzky, Raphael Götzky Raphael Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg DNA-based Serumanalyse, Blase, DNA-basiert carcinoma Operative Medizin microsatellites Molecular serological diagnosis and urin analysis in transitional cell bladder cancer Für das Urothelkarzinom der Harnblase gibt es derzeit keine serologischen Marker und keine suffizienten nicht-invasiven Untersuchungsmethoden für eine Karzinomdiagnose anhand von Urinproben. In der vorliegenden Arbeit wurde mit der Methode der Mikrosatellitenanalyse (MSA) eine genetische Charakterisierung beim Urothelkarzinom der Harnblase durchgeführt, um Alterationen in der Serum-DNA und Urin-Sediment bei an einem Urothelkarzinom der Blase erkrankten Patienten nachzuweisen. Insgesamt wurden von 2001 bis 2003 frische Tumor-, Blut-, Serum und Urin-Sedimentproben von 117 im Universitätsklinikum Marburg an einem Urothelkarzinom der Blase operierten Patienten prospektiv asserviert. Mittels der Phenol-Chloroform Methode wurde die DNA aus Tumor und Lymphozyten des Blutes isoliert. Frei zirkulierende Serum- und Urin-Sediment DNA wurde mit dem Untersuchungs-Kit (Mini-Kit, Qiagen) isoliert. Die MSA wurde mit insgesamt 10 polymorphen Markern für die chromosomalen Regionen 5q, 8p, 9p, 9q 13q, 14q, 17p und 20q angewendet, um tumorspezifische Alterationen der Serum- und Urin-Sediment DNA zu identifizieren. Nach Amplifikation mittels PCR wurde allelic imbalance und loss of heterozygosity mit dem automatischen Laser Sequenzer (ALFexpress II, Amersham-Pharmacia Biotech) detektiert. 20 gesunde Kontrollen wurden mit denselben Markern untersucht. Es konnten Serum-DNA Alterationen in 77,7% (87/112) identifiziert werden. Unter Anwendung von denselben 10 Mikrosatellitenmarkern konnten tumorspezifische Urin-DNA Alterationen in 85% nachgewiesen und somit eine Tumordiagnose gestellt werden (64/75). Vier gesunde Kontrollen zeigten Serum-DNA Artefakte. Dies bedeutet eine Spezifität von 80%. Die höchste Frequenz von Serum-DNA Alterationen wurde für die chromosomale Region 9p mit 35% gefunden. Die anderen Chromosomen zeigten Serum-DNA Alterationen in 16 bis 26%. Bei den Urin-Proben zeigte die Region 9q mit 37% die häufigsten Alterationen. Die Identifikation von Serum-DNA Alterationen war assoziiert mit dem Tumorstadium (p = 0,008) und häufiger in schlecht differenzierten Tumoren (p = 0,005). 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