Clinical Chemistry 49:7
`1058 –1065 (2003)
`
`Molecular Diagnostics
`and Genetics
`
`DNA Integrity as a Potential Marker for
`Stool-based Detection of Colorectal Cancer
`Kevin A. Boynton,1 Ian C. Summerhayes,2 David A. Ahlquist,3 and
`Anthony P. Shuber1*
`
`Background: Molecular genetic analysis of DNA in
`patient stools has been proposed for screening of colo-
`rectal cancer (CRC). Because nonapoptotic cells shed
`from tumors may contain DNA that is less degraded
`than DNA fragments from healthy colonic mucosa, our
`aim was to show that DNA fragments isolated from
`stools of patients with CRC had higher integrity than
`DNA isolated from stools of patients with healthy
`colonic mucosa.
`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 com-
`pared with fragments obtained from colonoscopy-nega-
`tive 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 pro-
`liferation and apoptosis. An assay of fecal DNA integ-
`rity may be a useful biomarker for the detection of CRC.
`© 2003 American Association for Clinical Chemistry
`
`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.
`*Address correspondence to this author at: EXACT Sciences Corporation,
`63 Great Road, Maynard, MA 01754. Fax 978-897-3481; e-mail tshuber@
`exactsciences.com.
`Received in revised form March 4, 2003; accepted March 28, 2003.
`
`Colorectal cancer (CRC)4 accounts for 10% of all cancer
`deaths and is second only to lung cancer as a leading
`cause of cancer-related mortality in industrialized nations
`(1 ). An extensive body of work has been published
`demonstrating CRC progression along a series of predict-
`able neoplastic pathways (2 ). This sequential progression
`allows early diagnosis of CRC at a time when treatment
`has a high probability of being curative and may facilitate
`preventive intervention (3, 4 ).
`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 perfor-
`mance 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 –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 devel-
`oped 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
`
`4 Nonstandard abbreviations: CRC, colorectal cancer; GE, genome-equiv-
`alent(s); FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodam-
`ine; and DMSO, dimethyl sulfoxide.
`
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`Clinical Chemistry 49, No. 7, 2003
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`1059
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`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 pa-
`tients 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-effi-
`ciency PCR amplification and the presence of high-molec-
`ular-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 pres-
`ence of cancer. These observations suggest that DNA
`integrity may be a valuable CRC biomarker.
`
`Materials and Methods
`
`materials
`All capture probes and PCR primers were synthesized by
`Midland Certified Reagent Company. LA Taq® DNA
`polymerase was obtained from PanVera Corporation.
`Nucleotides were purchased from Promega Corporation.
`
`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 deliv-
`ered 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 (EX-
`ACT Sciences). After stools were homogenized, we cen-
`trifuged 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 subse-
`quently extracted with Tris-saturated phenol (Invitrogen),
`phenol– chloroform–isoamyl alcohol (25:24:1 by volume),
`and chloroform. Total nucleic acids were then precipi-
`tated (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 contain-
`ing 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. Se-
`quence-specific DNA fragments were purified from total
`nucleic acid preparations by oligonucleotide-based hy-
`brid 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 subse-
`quent 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 in-
`cubation, the mixture with hybridized sequences was
`diluted in sterile H2O and added to paramagnetic poly-
`styrene 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 continu-
`ously for 1 h at room temperature, after which beads with
`bound biotin-oligonucleotide-DNA complexes were iso-
`lated 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 ␮L of a low-salt buffer
`[0.1⫻ 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 Biochemi-
`cals) mixed with 1⫻ 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 ␮L 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 reac-
`tion 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 at http://www.clinchem.org/content/vol49/is-
`sue7/). 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
`
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`Boynton et al.: DNA Integrity as Marker in Stool-based CRC Detection
`
`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 examin-
`ing DNA integrity. Genistein (Indoline Chemical Co.), a
`promoter of apoptosis, was prepared in dimethyl sulfox-
`ide (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 ⫻
`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 ␮L of elution buffer, and 5-␮L
`volumes per reaction were used in DNA integrity analy-
`sis.
`
`PCR amplification for model system. The amplification reac-
`tions consisted of DNA from each fraction mixed with 1⫻
`PCR buffer with 1.5 mM MgCl2, 200 ␮M each de-
`oxynucleoside triphosphate, 300 nM PCR primers, and 5
`U of LA Taq polymerase. The mixtures underwent ther-
`mal 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 visu-
`ally inspected.
`
`Results
`differential pcr efficiencies in a clinical
`population
`Differences in the efficiencies of PCR reactions for stool
`DNA from CRC patients and disease-free individuals had
`been observed previously by our group (10 ). Differential
`amplification was seen when a 200-bp PCR product was
`amplified from K-ras target sequences captured from the
`stools of six patients. Band intensities for PCR products
`amplified from DNA captured from stools of CRC pa-
`tients were higher than the intensities for PCR products
`obtained from colonoscopy-negative individuals. This in-
`
`Fig. 1. DNA integrity analysis PCR amplification scheme.
`One forward primer and six reverse primers (A–F) were used to amplify six
`different fragment lengths (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, and 2.4 kb)
`from four different loci (APC, p53, BRCA1, and BRCA2; also see the online Data
`Supplement).
`
`(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 con-
`siderably 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 electropho-
`resed, 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 in-
`spected. These reactions were performed to determine
`whether the capture of target DNA was successful. Al-
`though independent PCR reactions were performed in
`this study, we are currently developing a multiplex PCR
`format.
`
`TaqMan analysis. TaqMan analysis was performed on an
`ABI 7700 thermal cycler (Applied Biosystems) with prim-
`ers against a 200-bp region of the APC gene. A probe
`labeled with 6-carboxyfluorescein (FAM) and 6-car-
`boxytetramethylrhodamine (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), 1⫻ PCR primers (5 ␮M),
`and 1⫻ TaqMan probe (2 ␮M; Applied Biosystems). We
`used 5 ␮L 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.
`
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`Clinical Chemistry 49, No. 7, 2003
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`1061
`
`creased band intensity suggested increased PCR reaction
`efficiency, and we hypothesized that it may be attribut-
`able to a greater amount of template available in the
`reaction.
`
`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 pa-
`tients. 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.
`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 de-
`tection 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 sup-
`ported the amplification of longer PCR products com-
`pared with those amplified from individuals who were
`disease-free (Fig. 3, lanes 1– 8).
`
`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 obser-
`vations 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 sepa-
`rate loci were amplified from captured DNA, a total of 24
`bands could have potentially been detected (DNA integ-
`rity 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
`
`Fig. 2. DNA integrity analysis is more efficient than PCR efficiency alone for differentiating CRC.
`Detection of a high-molecular-weight (1.8-kb) band, using a different reverse primer than the 200-bp reaction (see Fig. 1), was specific to those patients with cancer.
`Patients 1, 4, and 14 have CRC, whereas patients 2, 3, 5– 8, 10 –13, and 15 do not. Patient 9 has a carcinoid tumor of the ileum. M.W., molecular weight.
`
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`Boynton et al.: DNA Integrity as Marker in Stool-based CRC Detection
`
`Fig. 3. Application of the DNA integrity assay
`to a clinical population.
`Detection of multiple high-intensity high-molecular-
`weight bands in CRC patients. Examples of stool
`DNA profiles are shown from colonoscopy-negative
`patients (Normal Controls; lanes 1– 8) and from
`CRC patients (lanes 9 –15). Multiple reverse prim-
`ers were used to detect 2.4-kb, 1.8-kb, 1.3-kb,
`800-bp, 400-bp, and 200-bp fragments of the APC
`gene in these samples. See Fig. 1 for primer
`scheme and Data Supplement for sequence-spe-
`cific PCR primer sequences. M.W., molecular
`weight.
`
`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 sam-
`ples 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 associa-
`tion 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 ␮g 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 mono-
`layer 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 concen-
`tration as genistein concentration increased. Thus, DNA
`integrity analysis monitored changes in high-molecular-
`weight DNA concentration as apoptosis was induced in
`tissue culture cells.
`
`Discussion
`The colonic mucosa maintains surface crypt architecture
`integrity through a balance of programmed cell death and
`cell proliferation (15 ). The disruption of this balance by a
`shift away from regulated apoptosis to steadily decreas-
`ing degrees of apoptosis and increasing cell proliferation
`has been implicated as a factor leading to changes in
`colonic mucosa undergoing neoplastic transformation
`(16 ). Furthermore, p53 (17 ), APC (18 ), and K-ras (19 )
`mutations in CRC may be involved in the deregulation of
`apoptosis as well as causing an increase in proliferation.
`We previously observed a correlation between the pres-
`ence of disease and the band intensities of PCR products
`obtained from stools of CRC patient and hypothesized
`that this indicated the presence of high-molecular-weight
`DNA fragments that may originate from nondegraded
`sources in the colon (10 ). A method that provides quan-
`titative analysis of DNA integrity in patient samples may
`allow both noninvasive diagnosis and monitoring over
`time.
`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 re-
`sponse of the healthy mucosa to both acute and chronic
`environmental injuries. Bile acids, necessary for the emul-
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`Clinical Chemistry 49, No. 7, 2003
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`Fig. 4. Model system suggesting that DNA integrity analysis differentiates changes in DNA integrity during apoptotic induction.
`Human prostate carcinoma cells (DU145) were grown to confluency and then treated with several concentrations of the apoptosis inducer genistein. Lanes 1– 6 of each
`sample type represent different concentrations of genistein for 48 h: lane 1, control; lane 2, DMSO control; lane 3, 10 mg/L; lane 4, 30 mg/L; lane 5, 50 mg/L; lane
`6, 70 mg/L. Sample types: A, medium; B, nonadherent cells suspended in medium; C, monolayer. Multiple reverse primers were used to detect 2.4-kb, 1.8-kb, 1.3-kb,
`800-bp, 400-bp, and 200-bp fragments of the APC gene in these samples. See Fig. 1 for primer scheme and the Data Supplement for sequence-specific PCR primer
`sequences. M.W., molecular weight.
`
`sification 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 trans-
`formed 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 pro-
`grammed 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 de-
`tected 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 sam-
`ples, 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 aerodiges-
`tive 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 colonos-
`copy-negative individuals for a range of high-molecular-
`weight DNA fragments. Again we observed that the
`presence of high-molecular-weight fragments was an in-
`dicator of disease (present in 15 of the 27 CRC patients
`and 2 of the 77 colonoscopy-negative control group mem-
`bers).
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`1064
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`Boynton et al.: DNA Integrity as Marker in Stool-based CRC Detection
`
`We observed a highly significant relationship between
`the incidence of cancer and the presence of high-molecu-
`lar-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 no-
`napoptotic 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 me-
`dium 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 sensi-
`tivity 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 molec-
`ular 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 progres-
`sion.
`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 pa-
`tients 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 robust-
`ness 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.
`
`This work was supported by grants from the National
`Institutes of Health (RO1 DK 59400; Principal Investiga-
`tor, Dr. Ian C. Summerhayes) and the National Cancer
`Institute (RO1 CA 71680; Principal Investigator, Dr. David
`Ahlquist), EXACT Sciences (Maynard, MA), The Lahey
`Clinic (Burlington, MA), and The Mayo Foundation
`(Rochester, MN). The Mayo Foundation is a minor equity
`investor in EXACT Sciences. Dr. David A. Ahlquist holds
`no stock and has received no consulting fees. Dr. Ian
`Summerhayes has no affiliation with EXACT Sciences.
`A. Shuber and K. Boynton are employees of EXACT
`Sciences. We thank Dr. Monica Boyce for help in manu-
`script preparation. ExactorTM is a registered trademark of
`EXACT Sciences Corporation.
`
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