Untersuchungen zu präanalytischen Faktoren zur Gewinnung von zellfreier DNA in Urinen und Entwicklung einer einfachen Methode zur Verarbeitung großer Probenvolumen

Liquid biopsy has great potential to revolutionize molecular cancer diagnostics in the future. As a source of liquid biopsy, urine offers unique advantages compared to other liquids considering that it can be obtained non-invasively, in large quantities and independently by patients, without the need for a doctor’s visit. At present there has been a lack of standardized procedures that are necessary for implementation in everyday clinical practice. The field of pre-analysis currently receives too little attention. For result comparisons standardized procedures constitute an absolute prerequisite. Furthermore, they are the basis for reliable results of in the future. As EDTA is commonly used as an inexpensive preservative contradictory statements re- garding its efficiency can be found. In experiments of this study, it could be shown that ETDA, contrary to previous reports, only offers reliable protection of cfDNA fragments under slightly alkaline conditions. Furthermore, it was found that upscaling sample volume is effective without the presence of inhibitory substances. Moreover, a calculation model based on the measured cfDNA yield of this study provides information on the mutant allele frequency (MAF) at which upscaling is useful. Since reports suggest that the MAF in urine is significantly lower compared to blood plasma, it can be expected that upscaling even above 100 ml is advisable. Finally, three different cfDNA fragments were quantified in three exons of two genes in 32 study samples. The target sequences contained diagnostically relevant codons of non- small cell lung cancer. A high correlation of the quantity of the three fragments was demonstrated, indicating a non-random distribution pattern of cfDNA. This finding is in accordance with earlier reports pointing out epigenetic regulatory patterns which protect fragment ends from degradation. As these were found more frequently in cancer patients, it has been possible to detect presence of disease.