Trans-Renal Cell-Free Tumor DNA for Urine-Based Liquid Biopsy of Cancer

Abstract

Cancer biomarkers are a promising tool for cancer detection, personalization of therapy, and monitoring of treatment response or recurrence. “Liquid biopsy” commonly refers to minimally invasive or non-invasive sampling of a bodily fluid (i.e., blood, urine, saliva) for detection of cancer biomarkers such as circulating tumor cells or cell-free tumor DNA (ctDNA). These methods offer a means to collect frequent tumor assessments without needing surgical biopsies. Despite much progress with blood-based liquid biopsy approaches, there are limitations—including the limited amount of blood that can be drawn from a person and challenges with collecting blood samples at frequent intervals to capture ctDNA biomarker kinetics. These limitations are important because ctDNA is present at extremely low levels in plasma and there is evidence that measuring ctDNA biomarker kinetics over time can be useful for clinical prediction. Additionally, blood-based assays require access to trained phlebotomists and often a trip to a healthcare facility. In contrast, urine is a body fluid that can be self-collected from a patient’s home, at frequent intervals, and mailed to a laboratory for analysis. Multiple reports indicate that fragments of ctDNA pass from the bloodstream through the kidney’s glomerular filtration system into the urine, where they are known as trans-renal ctDNA (TR-ctDNA). Accumulating studies indicate that the limitations of blood based ctDNA approaches for cancer can be overcome by measuring TR-ctDNA. Here, we review current knowledge about TR-ctDNA in urine as a cancer biomarker approach, and discuss its clinical potential and open questions in this research field.

Urine biomarkers in cancer detection: A systematic review of preanalytical parameters and applied methods

“…6 Despite the lack of understanding about how DNA crosses the renal barrier, several studies have shown the presence of transrenal cfDNA in urine for different physiological and pathological conditions, such as pregnancy, cancer and inflammation. 5,6,8,10,11 Recent studies showed that cell-free nucleic acids and tumor DNA are shed into the circulation and pass the renal barrier to be excreted in urine. 12,13…”

“…5,6 The large size class, cellular DNA, originates from genital tract cells or cell debris shed into urine, while the small size class, cell-free DNA (cfDNA), mainly originates from glomerular filtration of blood cfDNA. [5][6][7][8] Cell-free and circulating nucleic acids in blood are the results of apoptosis and/or necrosis in actively proliferating healthy cells or tumor tissue. Apoptosis can be distinguished from necrosis based on the produced DNA fragment size.…”

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

“…The concentration of cell-free DNA in liquid biopsies, in particular such as urine, is usually low and varies considerably among different specimens and type of disease. 21 Taking into consideration the challenge of low-abundance cfDNA targets in liquid biopsies, 2,7 a major advantage of urine as a biological specimen for molecular diagnostics resides in the possibility of volume up-scaling, thereby increasing detection sensitivity. [23][24][25][26] The current investigation was devoted to the question if open-end increment of specimen F I G U R E 5 Urine cfDNA concentrations of 32 individuals (EGFR gene exon 21,119 bp) ranged from 121 to 19,049 copies/mL (median 1642, IQR 605-5388).…”

Tumor-Derived Exosomes and the Role of Liquid Biopsy in Human Papillomavirus Oropharyngeal Squamous Cell Carcinoma

“…Circulating tumor DNA, sometimes referred to as cell-free DNA, consists of nucleic acid fragments released by malignant cells via both passive (e.g., necrosis) and active (e.g., exocytosis) processes 19 . In the past several years, several institutions have developed robust experimental protocols to readily isolate ctDNA derived from HPV + OPSCC tumors in patients' plasma and urine 13,20–23 . Such protocols utilize quantitative polymerase chain reaction, digital droplet polymerase chain reaction, or capture-based next-generation sequencing to detect minute quantities of the highly conserved and oncogenic HPV E6 and/or E7 oncogenes 24 .…”

“…19 In the past several years, several institutions have developed robust experimental protocols to readily isolate ctDNA derived from HPV + OPSCC tumors in patients' plasma and urine. 13,[20][21][22][23] Such protocols utilize quantitative polymerase chain reaction, digital droplet polymerase chain reaction, or capture-based next-generation sequencing to detect minute quantities of the highly conserved and oncogenic HPV E6 and/or E7 oncogenes. 24 The purported advantages of ctDNA biomarkers in managing HPV + OPSCC include avoiding painful oropharyngeal biopsies, reducing operating room costs and utilization, using them as prognostic and predictive biomarkers, and improving the precision of diagnosis relative to computed tomography, magnetic resonance imaging, and position emission tomography for surveillance.…”