Impact of cell-debris and room-temperature storage on urine circulating tumor DNA from hepatocellular carcinoma

The use of urine circulating tumor DNA (uctDNA) for cancer screening suffers from limited understanding of preanalytical processing. This study evaluated the impact of cell debris and 7-day room temperature storage on the quality and yield of transrenal DNA. Archived urine specimens collected from five hepatocellular carcinoma (HCC) patients and two pregnant women carrying male fetuses were used to assess the impact of cell debris on urine cell-free DNA (ucfDNA) isolation as measured by quantitative PCR for Y-chromosome DNA, or HCC-associated mutation and methylation markers, and by capillary electrophoresis. Prospectively collected urine from 21 HCC patients was aliquoted after collection for paired immediate freezing versus 7-day room temperature storage followed by freezing for further analysis. Cell debris contained more Y-chromosome DNA than supernatant in three of the six urine specimens tested from pregnant women, suggesting that cell debris can be associated with 20.6% to 84.9% of transrenal ucfDNA. Ninety-five percent (20 of 21) of frozen and room temperature urine pairs had overlapping DNA size distribution. ucfDNA quantity determined by quantitative PCR for TP53, CTNNB1, TERT, and HCC-associated uctDNA markers were statistically similar between room temperature and frozen samples. This suggests no significant difference in DNA degradation between the groups. The association of transrenal ucfDNA with cell debris and uctDNA stability at room temperature is significant to further the understanding of transrenal uctDNA preanalytical handling for HCC screening.