Identifikation frei zirkulierender Tumor- DNA durch den Nachweis tumorspezifischer Mikrosatelliten-Alterationen im Serum

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 Charakteris...

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Bibliographic Details
Main Author: Götzky, Raphael
Contributors: Knobloch, R. von (Prof. Dr. med.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2014
Operative Medizin
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Table of Contents: 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.