Human Urine Contains Small, 150 to 250 Nucleotide-Sized, Soluble DNA Derived from the Circulation and May Be Useful in the Detection of Colorectal Cancer (2024)

Study Subjects and Specimens

Participants were recruited from the Great Lakes New England Clinical Epidemiological Center including University of Michigan, Dana-Farber Harvard Cancer Center, Henry Ford Health System, and University of Toronto. Cancer patients were enrolled from the surgical or oncologic serives (before initiation of chemo- or radiation therapy or surgery). Most “healthy-no known neoplasia” controls were subjects enrolled at the endoscopy suites, but had undergone a negative colonoscopy. Patient samples were obtained under institutional Division of Human Subjects Protection (IRB) approvals.

Urine Collection and Fractionation

Fresh collected urine was immediately mixed with 0.5 mol/L EDTA, pH 8.0, to a final concentration of 10 mmol/L EDTA to inhibit the possible nuclease activity in urine sample, and stored at −70°C. To isolate total urine DNA, frozen urine sample was thawed at room temperature, and then placed immediately in ice before DNA isolation. Thawed urine would be processed for DNA isolation within an hour.

To separate the supernatant and cell debris of freshly collected urine, total urine was centrifuged at 1500 rpm at 4°C for 10 minutes. The supernatant was collected on ice. The cell pellet was then washed briefly, with 1 ml of sodium citrate buffer (pH 3.0). The wash-off solution was collected by centrifugation at 13,000 rpm for 90 seconds and combined with the supernatant fraction. The pellet after centrifugation was collected as the cell debris portion of urine.

Serum Collection

Fifty ml of blood was collected in heparinized containers from informed and consenting subjects. The samples were permitted to sit at room temperature for a minimum of 30 minutes (and a maximum of 60 minutes) to allow the clot to form in the red top tubes, then centrifuged at 1300 × g at 4°C for 20 minutes. The serum was transferred to a polypropylene, capped tube, and frozen on dry ice.

DNA Isolation

Urine samples were mixed with 1.5 volume of 6 mol/L guanidine thiocyanate (Sigma, St. Louis, MO) by inverting eight times. One ml of resin (Wizard DNA Isolation Kit, Promega, Madison, WI) was added into the urine lysate and incubated for 2 hours at room temperature with gentle mixing. Resin-DNA complex was centrifuged, transferred to a mini-column (provided from the kit), washed with a buffer provided by the manufacturer, and DNA was eluted with H₂O. Serum DNA was isolated as was the urine DNA, except 2.5 volume of 6 mol/L guanidine thiocyanate was used. DNA from paraffin-embedded tissue sections was isolated using the MasterPure DNA Kit (Epicenter, Madison, WI) per manufacturer’s instruction.

Restriction-Enriched Polymerase Chain Reaction Detecting Codon 12 Mutation of K-ras Gene

The restriction-enriched polymerase chain reaction (RE-PCR) assay, illustrated in Figure 2A (see Figure 2), distinguishing wild-type and mutant K-ras sequences in DNA isolated from urine, serum, or tissue is described as below. Twenty cycles of Hot-start PCR were performed as following: DNA, equivalent to 0.2 ml of urine, with 1.5 mmol/L MgCl₂, 1X PCR buffer (Qiagen, Valencia, CA), 0.5 U Hot-start Taq (Qiagen), 200 μmol/L dNTP, 0.1 μmol/L primers: L-Ext (5′ GCT CTT CGT GGT GTG GTG TCC ATA TAA ACT TGT GGT AGT TGG ACC T 3′) and R-Ext (5′ GCT CTT CGT GGT GTG GTG TCC CGT CCA CAA AAT GAT TCT GA3′) was mixed, denatured at 95°C for 15 minutes and followed by 20 cycles of 94°C for 30 seconds, 52°C for 30 seconds, and 1 cycle of 72°C for 5 minutes. These first 20 cycles of PCR was used to amplify both wild-type and mutated DNA, but only to introduce an artificial BstNI site to the 5′ end of amplified product derived from wild-type DNA. After the first round of PCR, the amplified products were digested with BstNI (New England Biolabs, Beverly, MA) to eliminate amplified products derived from wild-type DNA. One of 20 of the digested product was then subjected to the second Hot-start PCR with the 1 μmol/L primers L (5′ ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT 3′) and R-Bst (5′ GTC CAC AAA ATG ATC CTG GAT TAG C 3′) at the following condition: 95°C for 15 minutes, 40 cycles of 94°C for 30 seconds, 56°C for 30 seconds and followed by 1 cycle of 72°C for 5 minutes. This second set of primers introduced a BstNI site to the 3′ end of amplified product derived from both wild-type and mutated templates serving as internal control for the BstNI diagnostic digestion after the second PCR. The amplified products (87 bp) of the second PCR were digested to completion with BstNI (as shown by disappearance of the 87-bp DNA fragment), and resolved through polyacrylamide gels. The appearance of the 71-bp DNA fragment after BstNI digestion [as illustrated in Figure 1A] is the evidence of the existence of K-ras mutated DNA. Each sample was assayed 2 to 3 times. The samples was scored as “positive” for K-ras mutation when the 71-bp DNA fragments appeared after the second BstNI digestion of the PCR products in 2 of 2 or 2 of 3 repeated assays. As validation controls, the standard reconstitution samples (as in Figure 2C) were included in each assay.

Cloning and Sequencing of Mutated K-ras DNA

The 71-bp fragment of the second BstNI-digested PCR product from the RE-PCR assay was identified by 2% agarose gel electrophoresis, and isolated from the gel by using the Perfectprep Gel Cleanup Kit (Eppendorf, Germany). The purified 71-bp fragment was then cloned into the pPCR-Script Amp SK (+) cloning vector by using the PCR-Script Amp Cloning Kit (Strategene, La Jolla, CA USA). The insert was determined by PCR with the T7 and T3 primers, with the annealing temperature 55°C, 30 cycles. The plasmid DNA containing insert was then isolated by using the Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA). The plasmid DNA was sequenced at the Nucleic Acid Facility, Thomas Jefferson University, Philadelphia, PA.

Determination of the Ratio of Mutated K-ras DNA to Total K-ras DNA in Low MW and High MW Urine DNA

Total urine DNA was resolved in a 2.5% agarose gel with a molecular weight marker, stained with ethidium bromide. The gel slices corresponding to DNA size at 1 kb and larger (as the high MW urine DNA fraction) or between 100 to 400 bp (as the low MW urine DNA fraction) were excised respectively. DNA was eluted from the gel slices by the MERmaid SPIN Kit (BIO 101, Vista, CA) per manufacturer’s specification.

To measure the ratio of the amplifiable mutated K-ras DNA to total K-ras DNA, a serial of twofold dilutions of eluted high and low MW urine DNA fractions were prepared, and subjected to the PCR with K-ras L-Ext and R-Ext primers to determine the relative amount of amplifiable K-ras templates in each fraction, and the RE-PCR to assay for the K-ras mutation, with the standard reconstitution samples. The relative copy numbers of K-ras amplifiable template was estimated by comparing it to the standard DNA control. The ratio of mutated K-ras DNA, total K-ras DNA, and the fold enrichment of K-ras mutated DNA was then calculated.

DNA Can Be Recovered from Human Urine and Occurs as Two General Size Classes

It has been demonstrated that urine contains DNA and suggested that a portion of this DNA comes from the bloodstream crossing the kidney barrier.12,13 To confirm this observation, nucleic acids from the urine of five individuals, were isolated and examined. The amount of nucleic acids in each sample was determined to be between 40 and 200 ng/ml using spectrophotometry (Figure 1A). The nucleic acids, isolated and concentrated from 15 ml of urine, were resolved by electrophoresis through 8% polyacrylamide gels, stained with ethidium bromide, and shown to resolve into two distinct populations with respect to size: (1) a heterogenous high molecular weight (MW) form that remained near the well (H, 1 kb and larger) and (2) a relatively uniform low molecular weight form (L) between 150 to 250 bp (bp) (Figure 1B).

To verify the nature of nucleic acid recovered from urine, DNase I and RNase A digestions were performed before electrophoresis. The results (Figure 1B) show that both high and low MW forms of the nucleic acid are RNase resistant, but DNase sensitive. They are thus considered to be DNA.

Comparison of DNA Derived from the Blood and Urine

DNA isolated from the circulation (serum) and urine was compared by electrophoresis through an 8% polyacrymide gel. Consistent with other reports,14,15 the serum contained fragmented DNA as indicated in Figure 1C (lane S), as resolved by electrophoresis. Although the relative abundances of the different size populations vary, the low MW (L) urine DNA (lane U) is represented by mostly 1 to 2 nucleosomal size fragments.

To investigate whether the low MW urine DNA is cell-free, total urine was resolved before DNA isolation, by low-speed centrifugation into two fractions: the supernatant and the cell debris, as described in the Materials and Methods section. Figure 1D shows that DNA recovered from total urine (T), the supernatant (Sup), and the cell debris (C), respectively, as resolved in an 8% polyacrymide gel. As predicted, both high and low MW DNA was recovered from the total urine (T). Interestingly, low MW urine DNA was mostly recovered in the supernatant (Sup) of urine. DNA derived from the cell debris pellet (C) contained high MW urine DNA. This result supports the hypothesis that the low MW urine DNA species is enriched for DNA that is cell-free, and the high MW urine DNA is mostly derived from the cells shed from the urinary tract.

Evidence that DNA in Urine Is, in Part, Derived from the Circulation

To further test the hypothesis that urinary DNA was, at least partially, derived from the circulation, the relative amounts of mutated K-ras proto-oncogene in different tissue compartments derived from an individual with a colorectal cancer (CRC) containing a codon 12 mutant K-ras sequence, was determined. DNA derived from malignant cells, as stated, can appear in the circulation.16,17,18 Since the mutant K-ras sequence would be expected to be in the tumor, but not in other tissue, its presence (or relative enrichment) in the small versus large species of urine-derived DNA would be evidence that the small DNA in the urine contained a contribution from the circulation.

To detect the codon 12 mutated K-ras genes present in small DNA fragments, a gel-based restriction-enriched PCR (RE-PCR)10 was modified to permit amplification of DNA of less than 300 bp in length, which includes the size of the smaller species recovered from urine. This approach is graphically outlined in Figure 2A. As validation controls, DNA was prepared from sources known to have either mutant (human adenocarcinoma “12Val” SW480 cells) or wild-type (human hepatoblastoma HepG2 cells) K-ras sequences, and subjected to PCR, with the results shown in Figure 2C.

DNA prepared from the urine of an individual with no diagnosis of cancer (Zu) contained no detectable levels of mutant K-ras (no appearance of a 71-bp fragment after BstNI digestion) under the conditions of amplification used, although wild-type K-ras was readily detected (Figure 2B). DNA was also prepared from tumor (AAt) and surrounding non-tumor tissue (AAadj), serum (AAs), and urine (AAu) obtained from an individual (AA) with a biopsy-confirmed diagnosis of CRC. Figure 2B shows that although DNA derived from the non-malignant tissue surrounding the tumor did not contain detectable mutant K-ras, mutant K-ras sequences are present (the 71-bp fragment after BstNI digestion is detected), above background levels (15 copies/50 ng DNA/reaction) in all other sources (tumor, serum, and urine) from the cancer patient. The absence of mutant K-ras in the non-tumor-derived solid tissue indicates that this individual does not carry the mutant sequence in the germ line, and its presence in the tumor is the evidence for a somatic mutation.

Since mutant K-ras sequences present in the tumor derived from individual AA would be expected to be present in the blood, detection of mutant K-ras in both of these tissue compartments (blood and tumor tissue) is not surprising. However, the presence of mutant K-ras sequences in the urine of samples from individual AA (Figure 2B) is consistent with the theory that DNA from the circulation, containing tumor-derived mutant K-ras, traveled to the urine. If the mutant K-ras sequence present in urine was derived from the tumor in the colon, the type of mutation detected in the urine and in the tumor should be identical. As described in the Materials and Methods section, the 71 bp of DNA fragment after the second BstNI digestion of the PCR products from the RE-PCR of the DNA from urine and tumor was cloned and sequenced to determine whether the mutation detected in urine is the same mutation that was found in the tumor. The DNA sequencing data clearly demonstrate that both the urine and the tumor of patient AA contain the 12 Val mutation in the K-ras sequence (data not shown).

The Small Size Class of DNA in the Urine Comes from, at Least in Part, the Circulation

As stated, the presence of mutant K-ras sequences in the urine could have either been the result of trans-renal passage of DNA from the circulation to the urine, or from cells resident in the urinary tract, which happen to have the same K-ras mutation. Since the larger DNA class detected in the urine might be excluded by kidney filtration, we had reasoned that the most likely population of DNA in the urine, which was derived from the circulation, would be the small size class (150 to 250 bp). In this case, relative to the larger (greater than 1 kb) species of DNA isolated from urine, the small, 150- to 250-bp size class of DNA isolated from the urine of the individual with the mutant K-ras-positive tumor would be expected to be enriched in abundance for mutant K-ras sequences. To test this hypothesis, large- and small-sized classes of DNA isolated from the urine of two CRC patients (AA and O), and one patient with an adenomatous polyps (Q), were recovered from gel slices, following resolution through 2.5% agarose gels. Urine DNA from these three patients had been tested, and found to contain the same K-ras codon 12 mutation as their corresponding tumor or polyp (data not shown). Each DNA fraction recovered from the gel slice was subjected to a determination of the ratio of mutated K-ras DNA to total K-ras DNA as described in Materials and Methods.

Briefly, to determine the copy number of total amplifiable K-ras DNA and mutant K-ras DNA in each fraction, serial twofold dilutions for each fraction were prepared, and then subjected to (1) the PCR amplification for the total amplifiable K-ras DNA, and (2) the RE-PCR assay for the K-ras mutation, as summarized in Table 1. Analysis of the ratio of mutated K-ras DNA to total K-ras DNA in each size fraction (fold enrichment of mutated K-ras detected) shows a clear enrichment (8 to 12-fold) of mutated DNA in low MW DNA fraction for all samples tested. Since the separation of low MW DNA from high MW DNA is not expected to be absolute by agarose gel electrophoresis, it is not surprising that some amount of mutant K-ras would be detected in higher molecular species. However, it is reasoned that the mutated K-ras DNA detected in the low MW species isolated from urine comes from the circulation.

Detection of Mutant K-ras in Urine and Disease Tissue from the Patients with a Diagnosis of Either CRC or Adenomatous Polyps

Having established necessary assay parameters, the comparison between mutant K-ras in colorectal disease tissue (CRC or adenomatous polyps), and corresponding urine from the same individual was investigated. Paraffin-embedded disease tissue sections, as well as corresponding urine from five subjects with a diagnosis of CRC, and 15 subjects with the diagnosis of adenomatous polyps were collected according to the approved IRB protocol. DNA from the collected samples was isolated and subjected to the RE-PCR assay for the mutant K-ras sequence (as in Figure 2). As the control, DNA isolated from urine of 48 subjects with no known neoplasia (healthy) was also subjected to mutant K-ras detection by the RE-PCR assay. As shown in Table 2, 9 of the 48 (19%) urine samples from “healthy” individual contained detectable mutated K-ras sequences. Interestingly, all 9 positive samples were derived from the subjects enrolled at the endoscopy suites, but had undergone a negative colonscopy, as mentioned in Materials and Methods. Significantly, in each of five CRC samples shown to contain mutant K-ras, as shown in Figure 2D, mutant K-ras was detected in the corresponding urine (100% concurrence). Similarly, for 10 of the 13 polyps samples shown to contain mutant K-ras, mutant K-ras was detected in the corresponding urine (77% concurrence).

It is noted that mutated K-ras was detected in urine from one subject with polyp tissues containing no detectable mutant K-ras. Taken together, mutant K-ras sequences were detected in the urine of 15 of the 18 urine samples (83.3%) from individuals with confirmed mutant K-ras containing tumors or polyps, and 19% of those with no diagnosis of colorectal disease.

Supported, in part, by The Early Detection Research Network (EDRN) of The National Cancer Institute, The Hepatitis B Foundation of America, and an appropriation from The Commonwealth of Pennsylvania.

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