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Technical Briefs |
Departments of
1
Pathology,
2 Internal Medicine, and
3
Surgery, Canisius Wilhelmina Hospital, NL 6532 SZ Nijmegen, The Netherlands
4 Department of Surgery, University Medical Center St. Radboud, NL 6500 HB Nijmegen, The Netherlands
aaddress correspondence to this author at: Department of Pathology C66, Canisius Wilhelmina Hospital, Weg door Jonkerbos 100, NL 6532 SZ Nijmegen, The Netherlands; fax 31-24-365-8844, e-mail c.klaassen{at}cwz.nl
Analysis of nuclear DNA extracted from stool specimens (1) is a recent addition to cancer diagnostics (2)(3)(4). Most studies have focused on the detection of sequence variations in tumor suppressor genes and oncogenes and on their correlation with clinical stage. In addition, however, the amount of human DNA in feces may be increased in individuals with colorectal cancer. Villa et al. (5) found that ß-globin sequences were amplified by PCR more frequently in patients with either colorectal carcinoma or adenomas than in healthy individuals. Ahlquist et al. (6) demonstrated that large DNA fragments were amplified from DNA in stool samples from colorectal cancer patients more frequently than from healthy volunteers. In view of these results, we developed a real-time PCR assay for quantification of human DNA in stool samples.
Human stool samples were collected from 15 healthy adult volunteers (mean age, 46 years; range, 21–78 years) not on any dietary restrictions or antibiotic treatment and from 13 patients (mean age, 71 years; range, 49–81 years) who were diagnosed with colorectal cancer. All volunteers and patients gave informed oral consent. Stools were collected before any preparation for colonoscopy. Absolute care was taken to avoid hydration of the samples until further processing (1). Under these circumstances, solid stool samples can be stored at 4 °C for several days without significant degradation of the DNA. All stool samples from both groups were processed within 48 h after collection.
DNA was isolated from 200-mg fresh solid human stool samples with use of the QIAamp DNA Stool Mini Kit essentially according to the manufacturer’s recommendations for human DNA extraction. The purified DNA was eluted in 200 µL of elution buffer. The total yield of DNA was determined by ultraviolet absorbance at 260 nm on a DU 7600 spectrophotometer (Beckman); the A260/A280 ratio was 
1.8.
We targeted a 71-bp fragment within exon 3 of the human ß-globin gene and used a combination of PCR primers and TaqMan probe to amplify and detect only human DNA sequences. We selected human DNA sequences that included several mismatches with respect to known ß-globin gene sequences of edible species in the 3'-end region of both PCR primers and in the TaqMan probe. Primer and probe sequences were as follows: forward primer, 5'-GGGCAACGTGCTGGTCTG-3'; reverse primer, 5'-AGGCAGCCTGCACTGGT-3'; TaqMan probe, 5'-FAM-CTGGCCCATCACTTTGGCAAAGAA-TAMRA-3' (where FAM is 6-carboxyfluorescein, and TAMRA is 6-carboxytetramethylrhodamine). The specificity of the primers and probe was verified by BLAST analysis (7).
For PCR, a LightCycler (Roche) reaction mixture consisted of 1x FastStart Master Mix for Hybridization Probes (Roche), 250 nM each PCR primer, 100 nM TaqMan probe, 3 mM MgCl2 (total concentration), and up to 500 ng of total DNA in a reaction volume of 20 µL. PCR conditions were as follows: 10 min at 95 °C followed by 50 cycles of 0 s at 95 °C and 15 s at 60 °C, with maximum heating and cooling settings (20 °C/s). In each cycle, the fluorescent signal from the liberated FAM reporter group was measured in channel 1 at the end of the primer extension phase. The amounts of human DNA in the samples were extrapolated, using the "second derivative maximum" method, from a calibration curve constructed with fixed amounts of human placenta DNA. All quantification experiments were performed in triplicate. The technician performing the quantification experiments had no knowledge of the origin of the samples.
Stool samples (200 mg) from healthy adult volunteers contained a mean (SD) of 20 (12) µg of DNA (n = 15; total fecal DNA determined by ultraviolet absorbance). The relative concentration of human DNA (determined by real-time PCR) was 0.9 (0.6) mg/g of total fecal DNA (n = 15; range, 0–2.2 mg/g) The mean SE for replicate measurements of individual samples was 20%. Real-time PCR could have underestimated DNA if the final samples contained inhibitors of the real-time PCR process. We tested for PCR inhibitors by adding a fixed amount of human lymphocyte DNA to the fecal DNA samples before assay. When we analyzed 500 ng of total fecal DNA + 10 ng of human lymphocyte DNA, the samples contained, on average, 10.3 ng of human DNA (range, 7.8–16.6 ng). When we analyzed 500 ng of total fecal DNA alone, the average sample contained 0.45 ng of human DNA. From the 10 ng of human lymphocyte DNA that was added, we recovered, on average, 98.5%, demonstrating the absence of PCR inhibitors in the final DNA samples and the accuracy of the method.
A cutoff value of 2.7 mg of human DNA/g of total DNA was established (mean + 3 SD for samples from volunteers). We obtained positive results for six of nine samples from patients with left-sided colorectal cancer and for none of four samples from patients with right-sided colorectal cancer (
2 = 4.7; P <0.05; Table 1
).
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Using this approach, we demonstrate that stool samples from colorectal cancer patients contain increased concentrations of human DNA. Although based on a small number of samples, this justifies further investigation of this approach for detection of colorectal cancer. The increased concentrations of human DNA could be explained by decreased apoptosis of bowel cells and/or increased shedding of cancer cells and/or inflammatory cells into the colonic lumen. However, if the latter were the case, these results could also be obtained with benign pathologies of the colon. Such conditions remain to be investigated but could very well be studied by this approach.
No positive test result was obtained with patient samples with a tumor in the proximal part of the colon. An explanation for this may be the relative state of hydration in this part of the colon. A more hydrated environment is more hostile toward DNA with respect to nucleases and other hydrolyzing enzymes. Because the relative state of hydration decreases toward the more distal parts of the colon, a more favorable environment will be created for preservation of the DNA.
In this study, there may have been a confounding age difference between the patient group and controls. The mean age of the patient group was higher than that of the control group. However, within the patient group we demonstrated a difference between left-sided and right-sided colorectal tumors with no age difference being present. We therefore believe that this does not play a critical role in this study.
Usually, the amount of DNA in feces is calculated in absolute values (in mg of DNA/g dry weight). To compensate for differences in hydration states, the dry weight must be determined, for example, by freeze-drying. Although ideal for preservation of the DNA, freeze-drying is time-consuming and laborious and not available to every laboratory. Another complicating factor has been the need for quantitative extraction of human DNA from any amount of stool sample. We found, however, that all DNA (regardless whether it is of host, microbial, or food origin) that is liberated on lysis of the samples is further purified with equal efficiency (unpublished observations). We therefore quantified the amount of human DNA relative to the amount of total DNA. Thus, the amount of feces analyzed would be irrelevant, simplifying the entire procedure. Although stool samples are heterogeneous in composition, sampling errors introduced by analyzing small fractions may be circumvented by analyzing multiple fractions from each stool sample.
In conclusion, we describe a quantitative assay to determine the relative amounts of human DNA in feces. This approach allowed the detection of increased concentrations of human DNA in stool samples from patients with colorectal tumors, making this noninvasive assay a simple and potentially interesting approach to colorectal cancer screening.
References
The following articles in journals at HighWire Press have cited this article:
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  J. C. Mathers and J. E. Hesketh The Biological Revolution: Understanding the Impact of SNPs on Diet-Cancer Interrelationships J. Nutr., January 1, 2007; 137(1): 253S - 258S. [Abstract] [Full Text] [PDF]
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 W. K. Leung, K.-F. To, E. P.S. Man, M. W.Y. Chan, A. H.C. Bai, A. J. Hui, F. K.L. Chan, J. F.Y. Lee, and J. J. Y. Sung Detection of Epigenetic Changes in Fecal DNA as a Molecular Screening Test for Colorectal Cancer: A Feasibility Study Clin. Chem., November 1, 2004; 50(11): 2179 - 2182. [Full Text] [PDF]
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J. B. de Kok Quantification and Integrity Analysis of DNA in the Stool of Colorectal Cancer Patients May Represent a Complex Alternative to Fecal Occult Blood Testing Clin. Chem., December 1, 2003; 49(12): 2112 - 2113. [Full Text] [PDF]
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