Cell‐free DNA extraction from urine of lung cancer patients and healthy individuals: Evaluation of a simple method using sample volume up‐scaling

Abstract

BackgroundUrine holds promise as a source for cell‐free DNA (cfDNA) analysis of cancer genetics due to its nature as a self‐collectable biospecimen available in large quantities. However, pre‐analytical variables such as preservation of cfDNA or efficiency of up‐scaling specimen volume need to be better explored to increase analysis sensitivity.Patients and MethodsInitially effects of pH levels on cfDNA stability of urine preserved with EDTA were investigated in three healthy probands. Furthermore, the efficiency of urine volume up‐scaling was examined using a simple DNA extraction method and cfDNA in urine of 32 individuals. Quantification was performed by PCR detection of three relevant targets used for EGFR and KRAS gene mutational analysis.ResultsOnly samples preserved with EDTA at alkaline pH levels showed cfDNA stability of up to 10 days at room temperature. Moreover, it was found that increasing the volume up to 100 mL was highly efficient. A similar amount of copies was detected in three different gene sites in all specimens indicating both a good availability and a non‐random distribution pattern across genes. Since the median cfDNA copy number was 1642 copies/mL, abundance of cfDNA in this type of liquid biopsy is low.ConclusionPredictable sensitivities with different urine volumes on the ground of detectable cfDNA in our study population revealed that up‐scaling (>5 mL) is mandatory if the mutation allele frequency is less than 1%.

The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples

“…cfDNA in urine has shown great potential in noninvasive diagnosis. Urine is an ideal biomaterial for the study of urological diseases due to its direct contact with the urinary system and convenient collection of sufficient volume ( Ruppert et al, 2023 ). Research has shown significantly higher levels of urine cfDNA (ucfDNA) in patients with urinary tumors compared to healthy individuals ( Nikanjam et al, 2022 ).…”

“…Research has shown significantly higher levels of urine cfDNA (ucfDNA) in patients with urinary tumors compared to healthy individuals ( Nikanjam et al, 2022 ). Nevertheless, the clinical application of ucfDNA is relatively rare, possibly due to its sensitivity to environmental conditions (e.g., temperature and pH level of preservation solution) ( Kim et al, 2023 ; Ruppert et al, 2023 ), which makes it easier to degrade rapidly, resulting in inadequate concentrations for downstream analysis ( Nel et al, 2023 ) compared to blood-derived cfDNA. Therefore, the weaker stability makes ucfDNA analysis more susceptible to complex preanalytical variables, highlighting the importance of establishing a consensus on ucfDNA experimental procedures.…”