利用尿液游离DNA作为综合基因组分析的信息性生物材料

Background: Urinary cell-free DNA (UcfDNA) could soon prove to be a valuable non-invasive sampling analyte for next-generation sequencing (NGS) assays, particularly in urological cancers. Additionally, enabling at-home collection for cancer testing could lead to higher rates of adherence to testing schedules. In this proof-of-concept study, we intended to establish a baseline for how to effectively extract UcfDNA from urine with the potential of applying it as a sample type in assays such as Labcorp Plasma Complete TM . Methods: Extraction efficiency was compared across 4 different on-market cfDNA extraction methods, 3 manual kits (1 bead-based, 1 column-based, and 1 column/bead-based hybrid) and 1 automated bead-based kit. This comparison used 5 mL urine samples (n=10) that were stored at room temperature for 8-25 months. All samples were collected from patients with known solid tumor cancers. Extracted UcfDNA was characterized using both a fluorometer and a cfDNA bioanalyzer with a pre-determined cfDNA base pair range (50-700 bp). Next, larger volume (15 mL) extractions were conducted using 2 of the 4 previously tested extraction kits (the column/bead-based hybrid kit and automated bead-based kit) utilizing samples (n=6) collected with a urine stabilizing additive and stored at -80°C for 9-20 months. These extractions produced sufficient UcfDNA and were assessed for comprehensive genomic profiling in duplicate using Labcorp Plasma Complete. Post sequencing quality metrics were analyzed to determine the performance and reliability of the data. Lastly, mutation profiles were assessed for reproducibility between replicates and across extraction methods. Results: The results show that multiple urine extraction methods produce viable yields with significant variability (2 - 900 ng) and these were influenced by cancer type and pre-analytical factors such as sample handling and storage. Regarding post-sequencing quality metrics, the extraction kits had little impact. However, the quality of material extracted from each kit was variable, indicated by differences in sequencing ratios. Comprehensive genomic profiling results indicate robust performance, with a > 90% call rate when identifying variants > 0.8% variant allele frequency (VAF) across replicates. The assay also identified multiple variants with 100% call rate across sample replicates and extraction methods, including clinically significant variants such as a TERT promotor mutation and an ALK translocation. Conclusions: The findings show that UcfDNA is an extractable biomaterial when properly handled, and it also has great potential to be used for comprehensive genomic profiling in urological cancer samples with Labcorp NGS assays such as Labcorp Plasma Complete.