DNA in Urine
Although serum/plasma is the traditional bodily fluid used for monitoring circulating DNA levels, several studies have shown that urine may be used as well (Botezatu et al. 2000; Fitzgerald et al. 1995; Zhang et al. 1999). However, until recently, it was assumed that DNA found in the urine likely originated in the urinary tract from normal cell death in the kidneys, bladder, and associated tissues. In 2000, Botezatu et al. published a report on the detection of urinary DNA sequences that were different from the subjects' bulk nuclear DNA. More specifically, urine samples from women who received blood transfusions from male donors and from pregnant women with male foetuses were found to contain male-specific DNA sequences. Moreover, the urine of colorectal cancer patients was found to contain DNA with K-ras mutations that matched the K-ras mutations found in their tumours (Botezatu et al. 2000). In these cases, the DNA undeniably originated from outside of the urinary tract and passed through the kidney barrier. It was of high enough molecular weight to be analyzed by PCR and in-situ hybridization. This provided the first evidence that the kidney barrier is permeable to cell-free DNA and provided a direct correlation between plasma DNA and urine DNA. Urine contains DNA that originates outside of the urinary tract, travels through the blood stream, and crosses the kidney barrier (known as transrenal DNA). Therefore, urine could provide a completely non-invasive source of material to detect and monitor for cancer types other than those of the genitourinary system, to evaluate the effectiveness of therapy, and to detect relapses of the disease.
Muller et al., 2003 reported that carcinomas of the urinary bladder, renal cells, and prostate can be diagnosed from material obtained in urine samples. Cell-free DNA found in urine has already been associated with urinary tract cancer for some time, and in recent years this has been applied to increase the accuracy of tumour detection in the bladder and prostate (Fitzgerald et al. 1995; Goessl et al. 2000; Utting et al. 2002).
In 2004, Su et al. confirmed the presence of tumour-related DNA in urine, and expanded on this finding in a blinded study involving 20 patients with either colorectal carcinoma or adenomatous polyps. Mutant K-ras sequences were detected in the urine of 83.3% of patients who had confirmed K-ras mutations in their tumours or polyps, and in 19% of healthy controls. In addition, Su et al. (2004) made the important discovery that DNA isolated from human urine resolves into two size categories: larger fragments (greater than 1 kb) originating mainly from within the urinary tract, and smaller fragments (approximately 150 to 250 bp) originating at least in part from the circulation. These smaller fragments, then, should be the focus of further research to correlate transrenal DNA with non-genitourinary cancers.
It is interesting to note that a 150-bp DNA fragment is equivalent to approximately 90 kDa, substantially larger than the molecular weight cut-off of the kidney glomeruli, which is about 70 kDa (Lichtenstein et al. 2001). Thus, the mechanism by which DNA crosses the kidney barrier is somewhat puzzling, although several hypotheses have been proposed. The strand-like nature of DNA offers one clue, and de Gennes (1999) has proposed that DNA may be able to passively go through open pores in lipid bilayers by inserting lengthwise, like thread through the eye of a needle. Lichtenstein et al. (2001) offer a thorough examination of this riddle, and speculate that serum amyloid P (SAP) may be involved in DNA clearance through the kidney barrier.
Unlike molecular diagnosis using circulating DNA, urinary DNA analysis is completely noninvasive, and larger volumes can be obtained with comparative ease. Additionally, the protein concentration of urine is more than 1000-fold lower compared to plasma, making DNA isolation and detection much easier (Botezatu et al. 2000). The concentrations of DNA in plasma and in urine of healthy volunteers are similar, around 2-100 ng/mL (Botezatu et al. 2000, Chen et al. 1996; Fournie et al. 1993; Leon et al. 1977). Although this comparison seems unlikely at first glance, Botezatu et al. (2000) suggest that the amount of DNA that passes through 1 mL of blood per day is much higher than the amount of DNA that accumulates in the urine during this time, due to the rapid turnover rate of cell-free DNA.
It seems reasonable to surmise that the cancer types that have already been linked to elevated levels of circulating DNA will also be linked to elevated levels of urinary DNA. However, research in the area of urinary DNA is still in its infancy, and much remains to be done. Cancers of the genitourinary tract, specifically bladder, prostate, and renal cancers, have been correlated to increased urinary DNA concentrations, as well as specific mutated sequences that can be found in urine (Goessl et al. 2001; Hoque et al. 2004; Utting et al. 2002; Zancan et al. 2005). As for transrenal DNA, in addition to the link to colorectal cancer discussed above, Botezatu et al. (2000) also found a correlation to pancreatic cancer. In this study, five out of eight pancreatic cancer patients were found to have K-ras mutations in their urine; however, no tumour tissue was available to verify the presence of the same K-ras mutations in the cancer cells. Nevertheless, the authors note that 80-90% of pancreatic carcinomas have K-ras mutations, so this finding may be significant (Botezatu et al. 2000).
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