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1 Applied Research Group, EXACT Sciences Corporation, Maynard, MA 01754.
2 Cell and Molecular Biology, R.E. Wise Research & Education Institute, Lahey Clinic, Burlington, MA 01805.
3 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905.
aAddress correspondence to this author at: EXACT Sciences Corporation, 63 Great Road, Maynard, MA 01754. Fax 978-897-3481; e-mail tshuber{at}exactsciences.com.
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Abstract |
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Methods: We purified DNA from the stools of a colonoscopy-negative control group and patients with CRC and examined the relationship between long DNA fragments and clinical status by determining stool DNA integrity, using oligonucleotide-based hybrid captures with specific target sequences in increasingly long PCR reactions (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, 24 kb). DNA fragments obtained from CRC patients were compared with fragments obtained from colonoscopy-negative individuals for length and/or integrity.
Results: DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group.
Conclusions: The presence of long DNA fragments in stool is associated with CRC and may be related to disease-associated differences in the regulation of proliferation and apoptosis. An assay of fecal DNA integrity may be a useful biomarker for the detection of CRC.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stool testing methods are noninvasive, do not require cathartic preparation of the colon, and can be performed on stools at a centralized testing facility. However, the promise of screening tools such as the fecal occult blood test has not been fully realized because of major performance limitations and low compliance rates. Although the fecal occult blood test has been available for some years and may have lowered CRC mortality to some degree (5), early-stage CRC is only poorly diagnosed by this method (6)(7).
Molecular genetic screening assays that test stool DNA examine the genetic composition of the colonic mucosa, which is exfoliated into the colon (8). Many molecular markers associated with colorectal carcinogenesis have been well characterized (9) and have been shown to be represented in the DNA isolated from the stools of cancer patients (10)(11)(12). Previous work demonstrated that the use of multiple markers to screen patients for CRC is useful in detecting the heterogeneity of genetic lesions characteristic of CRC (8). A multitarget assay was developed to evaluate DNA isolated from human stools for 15 mutational hot spots in the K-ras, APC, and p53 genes, as well as for microsatellite instability in the BAT-26 locus (10). During the course of this study, it was observed that stool DNA from a colonoscopy-negative control group and CRC patients exhibited various efficiencies in PCR amplification, with DNA prepared from stools of CRC patients consistently amplifying more efficiently. It was postulated that differences in PCR efficiency may be related to the presence of a greater concentration of tumor-associated human DNA in the stools of CRC patients shed from lesions. Studies have demonstrated greater shedding of nonapoptotic colonocytes exhibiting cell surface tumor markers from CRC tumors into the mucocellular layer compared with shedding from healthy mucosa (13).
We have observed an association between high-efficiency PCR amplification and the presence of high-molecular-weight DNA fragments in the stool. Additionally, we observed that the presence of high-molecular-weight DNA fragments in the stool is associated with the presence of cancer. These observations suggest that DNA integrity may be a valuable CRC biomarker.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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studies on clinical specimens
Stool collection.
We consecutively recruited 25 patients with CRC and 77 patients with negative colonoscopic findings at the Mayo Clinic (Rochester, MN). All stools were collected before colonoscopy or purgative bowel cleansing. To prevent artifacts from toilet water, stools were collected in toto by use of a plastic bucket-type container mounted to the toilet seat and were then delivered to an onsite processing laboratory within 12 h of defecation. On receipt, stools were immediately aliquoted into 30-g portions, frozen at -60 °C, and later sent (at least 30 g per participant) on dry ice by 24-h express mail to EXACT Sciences laboratories (Maynard, MA) for analysis.
Stool homogenization.
All stool samples were thawed at room temperature and homogenized in an excess volume (7 mL of buffer per gram of stool) of EXACT buffer A (EXACT Sciences) using an ExactorTM stool shaker (EXACT Sciences). After stools were homogenized, we centrifuged a 4-g stool equivalent of each sample to remove all particulate matter and incubated the supernatants at 37 °C after addition of proteinase K (0.5 g/L) and sodium dodecyl sulfate (5 g/L). The supernatants were subsequently extracted with Tris-saturated phenol (Invitrogen), phenol–chloroform–isoamyl alcohol (25:24:1 by volume), and chloroform. Total nucleic acids were then precipitated (1/10 volume of 3 mol/L sodium acetate and an equal volume of isopropanol), removed from solution by centrifugation, and resuspended in Tris-EDTA [0.01 mol/L Tris (pH 7.4), 0.001 mol/L EDTA] buffer containing RNase A (2.5 mg/L), incubated for 15 min at 37 °C, and then stored at -20 °C. For each group of samples prepared, we included process positive-control samples as well as component negative controls.
Sequence-specific hybridization and selective capture.
Sequence-specific DNA fragments were purified from total nucleic acid preparations by oligonucleotide-based hybrid captures as described previously (10). In this method, DNA was combined in an equal volume of a solution of 6 mol/L guanidinium isothiocyanate with biotinylated oligonucleotide capture probe sequences (33 pmol; Midland Certified Reagent Company) that were complementary to the regions of the DNA fragments that were outside of the regions that were amplified in subsequent PCR amplification reactions. The mixture was heated to 95 °C for 5 min, incubated on ice for 5 min, and then incubated at room temperature overnight to allow for hybridization of complementary sequences. After incubation, the mixture with hybridized sequences was diluted in sterile H2O and added to paramagnetic polystyrene beads coated with streptavidin (Dynabeads® M-280 Streptavidin; Dynal ASA), which had been freshly washed according to the manufacturer’s instructions. The sample-bead suspension was rotated gently and continuously for 1 h at room temperature, after which beads with bound biotin-oligonucleotide-DNA complexes were isolated with a magnetic separator and washed with capture binding and washing buffer [1 mol/L NaCl, 10 mmol/L Tris (pH 7.2), 1 mmol/L EDTA, and 1 mL/L Tween® 20]. The captured sequences were then eluted from the bead complexes by the addition of 40 µL of a low-salt buffer [0.1x Tris-EDTA; 1 mmol/L Tris (pH 7.4), 100 mmol/L EDTA] and then heating the bead complex to 85 °C for 4 min. After removal of the beads, eluted sequences were stored at -20 °C.
PCR amplification of stool DNA samples.
Amplification reactions consisted of captured human stool DNA or human genomic DNA (Boehringer Mannheim Biochemicals) mixed with 1x PCR buffer with 1.5 mM MgCl2, 200 mM each deoxynucleoside triphosphate (dATP, dGTP, dTTP, and dCTP), 300 nM PCR primers, and 5 U of LA Taq polymerase. We used 5 µL of captured DNA in the PCR reactions. Although DNA concentrations varied, TaqMan® analysis indicated that, on average, each sample contained 114 genome-equivalents (GE); thus, each reaction contained 
800 pg of DNA. DNA fragments for integrity analysis were amplified from four different loci: APC, p53, BRCA1, and BRCA2 (see Fig. 1
and the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol49/issue7/). Independent reactions were performed for the detection of the 400- and 800-bp and 1.3-, 1.8-, and 2.4-kb fragments as follows: thermal cycling began with an initial denaturation step (92 °C for 2 min) followed by 10 cycles of sequential DNA denaturation (92 °C for 30 s), primer annealing (58 °C for 1 min), and primer extension (68 °C for 3 min), and 30 additional cycles of 92 °C for 30 s, 58 °C for 1 min, and 68 °C for 2 min. These reactions were completed with a final primer extension step (68 °C for 7 min). Reactions performed on stool DNA had a PCR profile different from that for tissue DNA. Because considerably more DNA is available from tissue than from stool, fewer cycles were required. PCR products were stagger-loaded on an agarose gel (3% SeaKem agarose; BioWhittaker Molecular Applications) and electrophoresed, and the ethidium bromide-stained PCR products were visually inspected. The PCR reactions for the 200-bp fragment were performed for 40 cycles with the same conditions as above, and products were visually inspected. These reactions were performed to determine whether the capture of target DNA was successful. Although independent PCR reactions were performed in this study, we are currently developing a multiplex PCR format.
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TaqMan analysis.
TaqMan analysis was performed on an ABI 7700 thermal cycler (Applied Biosystems) with primers against a 200-bp region of the APC gene. A probe labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) was used to detect PCR product. Primer and probe sequences are given in the online Data Supplement.
Amplification reactions consisted of captured human stool DNA mixed with TaqMan PCR Universal Master mixture (Applied Biosystems), 1x PCR primers (5 µM), and 1x TaqMan probe (2 µM; Applied Biosystems). We used 5 µL of captured DNA in the PCR reactions. TaqMan reactions were performed as follows: thermal cycling began with a primer annealing step (50 °C for 2 min), followed by 1 cycle of DNA denaturation (95 °C for 10 min) and 40 cycles of sequential DNA denaturation (95 °C for 1 min) and primer annealing (60 °C for 1 min). The ABI 7700 unit detected amplification products with the FAM/TAMRA probe, and data used in the calculation of GE per reaction were provided.
model system
Tissue culture and apoptosis induction.
An established in vitro apoptosis induction cell culture system was used to determine whether this methodology was in fact examining DNA integrity. Genistein (Indoline Chemical Co.), a promoter of apoptosis, was prepared in dimethyl sulfoxide (DMSO) to a stock solution (10 000 mg/L) from which working concentrations of 10, 30, 50 and 70 mg/L were made. Human prostate carcinoma DU145 cells (American Type Culture Collection) were seeded at a density of 2.5 x 105 cells per plate in DMEM supplemented with 50 mL/L fetal calf serum, 15 mmol/L HEPES, 4.5 g/kg glucose, 65 mL/L horse serum, 100 kilounits/L penicillin, and 100 g/L streptomycin. Twenty-four hours post-seeding, cells were exposed to a range of genistein concentrations and maintained for 48 h in the presence of this agent. Controls received no DMSO or genistein. DMSO controls received equal volumes of DMSO without genistein. At the end of treatment, attached cells were harvested by scraping, and cells in suspension were pelleted out of the medium to yield three distinct sample fractions: monolayer cells, detached cells, and medium containing cell-free DNA fragments. DNA was prepared from all fractions with the miniprep Qiagen blood DNA extraction reagent set. All samples were eluted in 200 µL of elution buffer, and 5-µL volumes per reaction were used in DNA integrity analysis.
PCR amplification for model system.
The amplification reactions consisted of DNA from each fraction mixed with 1x PCR buffer with 1.5 mM MgCl2, 200 µM each deoxynucleoside triphosphate, 300 nM PCR primers, and 5 U of LA Taq polymerase. The mixtures underwent thermal cycling that began with an initial denaturation step (92 °C for 2 min) followed by 10 cycles of sequential DNA denaturation (92 °C for 30 s), primer annealing (58 °C for 1 min), and primer extension (68 °C for 3 min); 16 (for lanes A1–B6 in the gel in Fig. 4
) or 26 (for lanes C1–C6 in the gel in Fig. 4
) additional cycles of 92 °C for 30 s, 58 °C for 1 min, and 68 °C for 2 min; and a final primer extension step (68 °C for 7 min). PCR products were stagger-loaded on an agarose gel and electrophoresed, and ethidium bromide-stained PCR products were visually inspected.
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Results |
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association of high-molecular-weight stool dna with the presence of crc
To further characterize the nature of the DNA fragments being obtained from these individuals, we determined fragment sizes. We determined the presence of high-molecular-weight template DNA by amplifying 200-bp and 1.8-kb target sequences within the APC gene, using stool DNA from cancer and colonoscopy-negative patients. Although we observed differential amplification, cancer and colonoscopy-negative samples both contained the 200-bp fragment. However, cancer-positive samples (Fig. 2
, lanes 1, 4, 9, and 14) had substantially greater amounts of the 1.8-kb fragment than samples from colonoscopy-negative patients (Fig. 2
, lanes 2, 3, 5–8, 10–13, and 15). Lane 9 represents PCR products amplified from stool DNA of a patient with a malignant ileal carcinoid tumor.
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With the correlation between the presence of the 1.8-kb fragment and cancer incidence, we wanted to further characterize DNA integrity in stools from colonoscopy-negative and cancer patients. We therefore developed a multiple-band high-molecular-weight DNA integrity detection scheme (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, and 2.4 kb). As seen in Fig. 3
, patients with CRC (lanes 9–15) contained high-molecular-weight human DNA sequences in their stool. This high-molecular-weight DNA supported the amplification of longer PCR products compared with those amplified from individuals who were disease-free (Fig. 3
, lanes 1–8).
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In addition, as observed with the 200-bp fragment in Fig. 3
, samples from colonoscopy-negative patients tended to have lower DNA concentrations compared with CRC samples. However, we have observed samples from cancer-free individuals with high DNA concentrations but low DNA integrity and have observed samples from CRC patients with low DNA concentrations and high integrity (see the online Data Supplement). These observations suggest that there is no direct association between total DNA concentration and integrity.
A DNA integrity scoring algorithm was developed based on the comparison of relative intensities of PCR products found in colonoscopy-negative individuals and CRC patients. Because six PCR products from four separate loci were amplified from captured DNA, a total of 24 bands could have potentially been detected (DNA integrity score, 0–24). Analysis of PCR products amplified from a 77-member colonoscopy-negative control group indicated that the majority of stool DNA from disease-free patients had scores between 0 and 18. We then analyzed the integrity of DNA from the stools of 27 patients with CRC. Using the previously developed algorithm, we found that 15 of 27 CRC patients had >18 bands, whereas only 2 of the 77 in the colonoscopy-negative control group had >18 bands (see the online Data Supplement). We compared total GE, as determined by TaqMan analysis using a 200-bp sequence, and DNA integrity scores. The left-hand column of Table 2 of the Data Supplement (http://www.clinchem.org/content/vol49/issue7/) has samples sorted by GE, whereas the right-hand column has the same samples sorted by DNA integrity score (total bands). When this data set was sorted by GE score and the specificity was set at 97%, sensitivity was 7%. When the sensitivity was set at 50%, data sorted by GE score yielded 77% specificity. When these same data were sorted by DNA integrity score and the specificity was set at 97%, sensitivity increased to 57%. When the sensitivity was set at 50%, data sorted by DNA integrity score yielded a 97% specificity. When samples were sorted by the number of bands (DNA integrity), there was a highly significant association between cancer and the presence of high-molecular-weight bands (P <0.001). When the same samples were sorted for GE, although there was a significant association between higher DNA concentration (200-bp band) and the incidence of cancer (P <0.01), the association between DNA integrity and cancer was stronger than the association between total human DNA concentration in a patient sample and cancer.
dna integrity analysis in a model system
To better understand the origin of the high-molecular-weight DNA fragments associated with the presence of CRC, we used our DNA integrity analysis method to examine the integrity of DNA generated from a tissue culture cell model exposed to the apoptosis promoter genistein. Subconfluent human prostate carcinoma DU145 cells (14) were exposed to 10, 30, 50, and 70 µg of genistein over a 48-h period. The 200-bp, 400-bp, 800-bp, 1.3-kb, 1.8-kb, and 2.4-kb DNA fragments were amplified from DNA isolated from adherent cells, detached floating cells in the culture medium, and cell-free DNA in the medium. As seen in Fig. 4
, neither the adherent monolayer cells (section C, lanes C3–C6) nor the cells in suspension (section B, lanes B3–B6) revealed a genistein dose-dependent response. High-molecular-weight DNA fragments were found in the monolayer cell fraction and may represent uncleaved DNA isolated from a largely nonapoptotic induced cell population. DNA from cells in suspension contained both high- and low-molecular-weight DNA fragments. This fraction may be a mixture of pyknotic fragments as well as nonapoptotic cells that have lost adhesion with the culture substrate. Conversely, the cell-free DNA amplified from the medium contained predominantly low-molecular-weight DNA fragments (Fig. 4
, section A, lanes A3–A6) that increased in concentration as genistein concentration increased. Thus, DNA integrity analysis monitored changes in high-molecular-weight DNA concentration as apoptosis was induced in tissue culture cells.
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Discussion |
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Adenomatous and neoplastic tissues along with healthy mucosa shed cells and/or cell fragments into the lumen of the colon (13). DNA isolated from stool samples is representative of the genetic composition of the colonic mucosa at the time of stool sample collection. Whereas the majority of mucosal tissues lose cells to apoptosis, there is evidence that transformed colonic mucosa cells have dysfunctional apoptotic mechanisms (16)(20) and thus may shed cells that have not undergone apoptosis.
The role of apoptosis is critical for the dynamic response of the healthy mucosa to both acute and chronic environmental injuries. Bile acids, necessary for the emulsification of dietary fats, have been shown to induce apoptosis in healthy colonic mucosa (21). Additionally, exposure to an excessively high-fat diet may lead to a colonic environment that is high in bile acids. It has been suggested that this may lead to the selection of transformed colonic mucosa that are insensitive to apoptotic induction by bile acids (22). It was hypothesized that as a result of the abrogation of apoptotic mechanisms in these cells, genetic damage that typically would signal programmed cell death is retained and may contribute to cancer progression in the colon (23).
Because one of the characteristics of apoptosis can be the cleavage of DNA into 180- to 210-bp fragments (24), the presence of high-molecular-weight fragments detected by the examination of DNA integrity in stools from cancer patients may indicate that a certain population of cells has escaped apoptosis-induced DNA degradation. Tumor cells that have escaped apoptosis and are necrotic may constitute many of the cells shed into the colonic lumen. One possible consideration is that the detection of longer DNA fragments in the stools of CRC patients may be the consequence of nuclease protection. The chromatin structure of the necrotic cells may not be degraded, and thus the DNA may not be available to nuclease-mediated cleavage.
To examine the relationship between cancer and the presence of high-molecular-weight DNA in patient samples, stools from patients with CRC and those with negative colonoscopies were analyzed for DNA integrity by the detection of 200-bp and 1.8-kb DNA fragments. Stools from CRC patients contained high-molecular-weight DNA fragments, whereas stools from patients who were negative by colonoscopy lacked the 1.8-kb band. These early results suggested that DNA integrity assessed in stool could accurately differentiate between disease-free individuals and CRC patients. Additionally, we observed that stool DNA from a patient with a malignant ileal carcinoid tumor, an aerodigestive cancer, contained high-molecular-weight DNA fragments. This observation suggests that DNA exfoliated from aerodigestive cancers may serve as a distinguishing diagnostic marker when recovered from stool, deserving further study.
We further examined the association between stool DNA integrity and the clinical status of the patient by analyzing stools from 27 CRC patients and 77 colonoscopy-negative individuals for a range of high-molecular-weight DNA fragments. Again we observed that the presence of high-molecular-weight fragments was an indicator of disease (present in 15 of the 27 CRC patients and 2 of the 77 colonoscopy-negative control group members).
We observed a highly significant relationship between the incidence of cancer and the presence of high-molecular-weight bands in patient samples. These data suggest that DNA integrity analysis is more informative in CRC detection than the determination of total human DNA in patient samples.
To determine the ability of this method to detect differences in DNA integrity between apoptotic and nonapoptotic cells, we applied our DNA integrity analysis to a model tissue culture system. DNA of cells from a prostate cancer culture cell line that had undergone apoptotic induction with genistein (14) was examined for the presence of high-molecular-weight DNA fragments. Observation of long PCR products may indicate retention of DNA integrity in those cell populations with lower apoptotic index (noninduced cellular monolayer). DNA prepared from substrate-adherent monolayer cells was enriched for longer fragments, cells floating in the medium contained both shorter and longer fragments, and the medium contained cell-free DNA of short fragment size. DNA integrity analysis thus provided a fragment length profile for these three cell populations, detecting changes in DNA integrity. These data illustrate the sensitivity of our method for the detection of shifts in DNA integrity. DNA integrity analysis, as demonstrated in our model system, may contribute to screening, staging, and monitoring of disease with use of cell-free DNA purified from bodily fluids in a variety of clinical settings.
Although current methodologies used to investigate the role of apoptosis in disease progression detect molecular components or byproducts of apoptotic induction, DNA integrity analysis may be a useful method for detecting the abrogation of apoptosis. Furthermore, this method may be useful for staging and monitoring of treatment in CRC and perhaps in other cancers, such as bladder (25), prostate (26)(27), and ductal carcinoma in situ (28), and in other diseases, such as inflammatory bowel disease (29), where abrogation of apoptosis has been indicated as an intrinsic factor in disease progression.
Because of the quantitative nature of this assay, it may be possible to adjust the sensitivity to apply it to the detection of abrogated apoptosis as a transition state. This transition state may be a characteristic of high-risk patients and may provide a means to differentiate between high- and low-risk individuals.
In efforts to apply the analysis of DNA integrity as a CRC screening modality, a scheme for quality assurance would be of considerable value. Some aspects of this scheme would include experimentation to test the robustness of intraassay performance, intraindividual assay outcome reproducibility, stability of DNA in samples, and analysis and optimization of sample collection, shipping, and storage conditions. Furthermore, it would be of great value to determine the performance characteristics of this assay by including it in a large clinical study that is representative of an appropriate target population.
In conclusion, the differential and quantitative analysis of stool DNA integrity may be a sensitive and specific biomarker useful for the detection of CRC and may be a powerful tool for detecting the presence of CRC.
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Acknowledgments |
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Footnotes |
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References |
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