Affiliations of authors: Department of Medicine and Ireland Comprehensive Cancer Center, Case Western Reserve University, and University Hospitals of Cleveland, Cleveland, OH (W-DC, JS, LM, PP, SL, JKVW); Exact Sciences, Marlborough, MA (JS, ZJH, JO, AS); Department of Pathology and Ireland Comprehensive Cancer Center, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH (DD, JW, TPP); Department of Epidemiology and Biostatistics and Ireland Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH (JSR); Department of Medicine and Ireland Comprehensive Cancer Center, Case Western Reserve University and University Hospitals of Cleveland, and Howard Hughes Medical Institute, Cleveland, OH (JL, LK, SDM)
Correspondence to: Sanford Markowitz, MD, PhD, Case Western Reserve University, WRB 3-127, 10900 Euclid Ave., Cleveland, OH 44106-7285 (e-mail: sxm10{at}cwru.edu).
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ABSTRACT |
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INTRODUCTION |
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Colon cancer is the second-leading cause of cancer death in adults in the United States (13). When these cancers are detected in early clinical stages, i.e., stages I and II, when the tumors are still confined to the bowel wall, surgical cure rates are 90% and 75%, respectively (14). In contrast, chances for cure drop rapidly once colon tumors have spread beyond the confines of the bowel. Initial reports have confirmed the potential for early detection of colon cancerderived aberrantly methylated DNA in both patient blood and feces, but the sensitivity and specificity of currently identified markers are not optimal (8,10).
To expand the population of genomic DNA sequences that might potentially be useful as methylated DNA markers of colon cancer, we have investigated whether cancer-associated aberrant DNA methylation might target CpG-rich sequences within a gene that is not expressed by normal colonic epithelium and for which gene silencing would therefore not result from an aberrant methylation event. We chose for this approach the vimentin gene, which encodes a protein constituent of intermediate filaments and whose expression is considered a classic marker of mesenchymal cells, such as fibroblasts (15), and which hence should not be expressed by normal colonic epithelium. We describe here the analysis of aberrant methylation of the human vimentin gene and then the assay of vimentin gene methylation as a potential marker of colon cancer in patient tumors and in fecal DNA.
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MATERIALS AND METHODS |
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Normal and malignant colon tissue samples were obtained from discarded tissue specimens from the department of surgical pathology at University Hospitals of Cleveland using a tissue procurement protocol approved by the University Hospitals of Cleveland internal review board. These samples included 12 samples of histologically normal colonic mucosa from individuals having resections for noncancer diagnoses (designated normal group 1) and 46 samples of histologically normal colonic mucosa from colon cancer resections (designated normal group 2), along with matching colon cancer tissue from these 46 patients (designated group A). An additional and independent set of 107 colon cancer tumor tissues (designated group B) were collected from consenting patients at the Lahey Clinic (Burlington, MA) and sent to Exact Sciences, which provided these samples for study at Case Western Reserve University. Colon cancer tumors included those arising in the proximal colon (cecum, ascending, and transverse colon), distal colon (descending and sigmoid colon), and rectum. VACO series colon cancer cell lines were established and maintained as described previously (16). For initial screening of vimentin gene methylation the 11 cell lines studied were Vaco5, Vaco6, Vaco9m, Vaco10m, Vaco206, Vaco241, Vaco364, Vaco394, Vaco400, Vaco425, Vaco441, and Vaco576. Additional studies also employed Vaco6. RNA and DNA were prepared from colon tissues and cell lines after lysis in guanidine isothiocyanate and fractionation through cesium chloride as previously described (17).
Immunohistochemistry
Vimentin protein expression in paraffin-embedded normal colon tissue and colon tumors were evaluated using a mouse anti-vimentin monoclonal antibody, V9 (DAKO Cytomation, Carpinteria, CA). Briefly, 5-µm sections of formalin-fixed, paraffin embedded-tissues were deparaffinized and rehydrated through graded alcohols to water. Antigen unmasking was performed by heat treatment (10 mM citrate, pH 6.0, in an 800-W microwave oven for two 5-minute cycles). Slides were incubated with the V9 anti-vimentin primary antibody at 1 : 100 dilution for 10 minutes and developed using the LSAB2 visualization system (DAKO) with 3,3' diaminobenzidine tetrahydrochloride substrate, followed by hematoxylin counterstaining. In every analysis, longitudinally cut sections of peripheral nerve were included as a positive control and staining with preimmune mouse serum was performed as a negative control.
Preparation of Colonic Mucosa and Colonic Crypts
Colonic mucosa was prepared by blunt dissection from normal portions of colectomy resections, with tissue maintained at 4 °C throughout. To further prepare colonic crypts, which are epithelial cell-enriched, mucosal samples were cut into 2- to 3-mm strips, incubated with approximately 5 mL of Cell Recovery Solution reagent (Becton Dickinson, Franklin Lakes, NJ) per square centimeter of tissue at 4 °C with gentle rocking for 1 hour, and then passed through a large-bore pipette. Released colonic crypts were collected by low-speed centrifugation at 350g for 5 minutes at 4 °C.
Real-time Reverse TranscriptionPolymerase Chain Reaction
The vimentin transcript was amplified from the isolated RNA of normal colon and colon cancer tissues and colon cancerderived cell lines in an iCycler instrument (BioRad Laboratories, Hercules, CA) using 400 nM of forward primer, 5'-CACGAAGAGGAAATCCGGAGC-3', and reverse primer, 5'-CAGGGCGTCATTGTTCCG-3', to yield a 215-bp product. Each PCR was carried out in a 25-µL volume using SybrGreen Mastermix (BioRad) for 8 minutes, 30 seconds at 95 °C, followed by 50 cycles of 95 °C for 20 seconds, 60 °C for 20 seconds, and 72 °C for 20 seconds. To directly compare vimentin expression in crypt cell preparations and in whole-colonic mucosa, vimentin transcript expression was normalized in both crypt and whole-mucosal preparations to the transcript levels of Muc2, a marker of colonocyte epithelial cell mass. Muc2 transcript was amplified using forward primer 5'-TGAAGAAGACAGAGACCCCCT-3' and reverse primer 5'-CAGGCAGTCCTCATTGTTCTGAC-3', spanning exons 14 and 15. The RT-PCR conditions were 50 cycles of 94 °C for 20 seconds, 60 °C for 20 seconds, and 72 °C for 20 seconds. The level of vimentin expression was determined as the ratio of vimentin to Muc2 = 2CTvimentin CTMuc2, where CTvimentin is the cycle number for crossing the iCycler detection threshold in real-time PCR amplification of vimentin, and CTMuc2 is the cycle number for crossing the iCycler detection threshold in real-time PCR amplification of Muc2.
Bisulfite Conversion of Genomic DNA and MS-PCR
Bisulfite conversion of DNA was performed as described previously (6,18) to create a template for methylation-specific PCR (MS-PCR). Briefly, 500 ng to 2 µg of genomic DNA from each sample in a volume of 50 µL was denatured by NaOH (freshly made, final concentration, 0.2 M) at 37 °C for 15 minutes. Next, 30 µL of 10 mM fresh hydroquinone and 520 µL of freshly prepared 3.0 M NaHSO3, pH 5.0 (Sigma, St. Louis, MO) were added, and the mixture was incubated at 55 °C for 16 hours. Bisulfite-modified DNA was purified using the Wizard DNA Cleanup kit (Promega, Madison, WI). The DNA was desulfonated by incubation with NaOH at a final concentration of 0.3 M at room temperature for 15 min and neutralized by adding ammonium acetate, pH 7.0, to a final concentration of 3 M. DNA was precipitated with ethanol and resuspended in distilled water to a final concentration of 5 ng/µL.
Bisulfite-treated DNA was then used as the template for MS-PCR, which was performed as described previously (6,18). Briefly, 5 µL of bisulfite-converted genomic DNA served as the PCR template. The amplification was in a reaction of 25 µL containing 0.19 mM each dNTP, 1.5 mM MgCl2, 400 nM of forward and reverse primers, and 1.25 U of AmpliTaq Gold in the recommended buffer. Amplification primers and reaction conditions are provided (Supplementary Table, available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97issue15). Vimentin MS-PCR reaction #29 employed forward amplification primer 5'-TCGTTTCGAGGTTTTCGCGTTAGAGAC-3' and reverse amplification primer 5'-CGACTAAAACTCGACCGAC TCGCGA-3'. PCR cycling parameters were as follows: hot start at 95 °C for 9 minutes, followed by 45 cycles of 95 °C (45 seconds), 70 °C (45 seconds), and 72 °C (45 seconds), then 72 °C for 10 minutes, and 10 °C to cool. For amplifications from fecal DNA, both forward and reverse MS-PCR primers were additionally extended by addition of a 5' tag sequence 5'-GCGGTCCC-3', which is not derived from the vimentin sequence but which provided, on the second and subsequent cycles of PCR, for more robust amplification of templates that had incorporated the PCR primers. For sequencing of bisulfite-converted DNA, products were amplified with methylation-indifferent primers and cloned into pCR2.1 TOPO TA cloning vector (Invitrogen, Carlsbad, CA); 1015 individual clones per sample were then sequenced using an automated sequencer (Applied Biosystems, Foster City, CA).
Preparation of Fecal DNA
Stools were collected from a population (n = 198) of average-risk individuals with no prior history of colon cancers or polyps and from a population (n = 94) of colorectal cancer patients, all of whom provided written informed consent, and who represented four different medical care organizations, of which one group contributed half of the total samples studied, with the remaining three groups contributing the balance. Stool samples were frozen within 72 hours after collection and stored at 80 °C. For recovery of human DNA, whole samples were thawed at room temperature and homogenized in excess volume (1 : 7) of EXACT buffer A (EXACT Sciences, Marlborough, MA). Homogenized samples were then archived at 80 °C for an average of 12 months (range = 618 months). No effect of the time of sample storage on ultimate sensitivity of the MS-PCR assay was found. To reduce the risk of sample degradation, homogenates were thawed only once, at the time of processing and analysis. At that time, a 4-g stool sample equivalent of each homogenate was centrifuged to remove all particulate matter, and the supernatants were treated with 20 µL of RNase A (2.5 mg/mL) (Roche, Indianapolis, IN) and incubated at 37 °C for 1 hour. Total DNA was then precipitated (by adding 1/10 volume of 3 M NaAc and an equal volume of isopropanol), and the DNA was resuspended in 4 mL of 1x TE buffer (0.01 M Tris, pH 7.4, 0.001 M EDTA) (Pierce, Rockford, IL). Target human vimentin DNA fragments were purified from total DNA preparations by acrylamide gelbased affinity capture as previously described (19). Total DNA yields from normal patients (median = 936 genome equivalents, range = 3318 560 genome equivalents) and cancer patients (median = 1014 genome equivalents, range = 323700 genome equivalents) were similar. Total captured DNA from each sample was then subjected to bisulfite-modification and MS-PCR, and the results were analyzed in a manner blinded to patient's disease status.
Statistical Analysis
Exact 95% confidence intervals (CIs) were calculated for all estimated proportions. Clinical variables were adjusted using a logistic regression model, and two-sided P values were calculated for the log-odds ratios using a Wald-type test (20). Comparisons were determined to be statistically significant if P<.05. MS-PCR reactions were run independently in quadruplicate on all cell line samples and in duplicate on all patient tissue samples. Due to limitations of sample amount, assays on aberrant crypt foci and on fecal DNAs were single determinations.
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RESULTS |
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Immunohistochemical assay of vimentin expression in the human colon showed the absence of protein expression in the colonic epithelial cells in both normal colonic crypts and in colon cancers and positive vimentin expression in stromal cells and lymphocytes within both normal colonic crypts and colon cancers (Fig. 1, A). To confirm that the vimentin gene is transcriptionally silent in colon epithelial cells, we used real-time RT-PCR to analyze vimentin transcript levels in bluntly dissected normal colonic mucosa, which contains epithelial and stromal cells and in a purified preparation of normal colonic crypts that are highly enriched for colonic epithelial cells (Fig. 1, B). On average, colonic crypts retained only 3% (95% CI = 2.9% to 3.1%) of the vimentin transcript level present in the full mucosal tissue (Fig. 1, C), strongly suggesting that vimentin transcripts in the normal mucosal tissue are derived essentially completely from the nonepithelial cell population.
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Acquired Increased Vimentin Methylation in Tissues From Primary Colonic Neoplasms
MS-PCR assays for vimentin gene methylation were next used to characterize vimentin gene methylation in matched pairs of normal colonic mucosa and colon cancer tissues obtained from 46 colon cancer patients not mentioned above. In this second set of 46 normal mucosal tissue samples, MS-PCR primer sets 3 and 29 again defined a 216-bp region of vimentin exon 1 that was devoid of any detectable methylation in 45 of 46 samples assayed (Fig. 2, D; Table 1). In contrast, 83% (38 of 46) of the colon cancers from the same 46 patients had acquired increased methylation in this 216-bp region, particularly when assayed by MS-PCR primer set 29 (Fig. 2, E, Table 1). Among these 46 colon cancers, acquired increased vimentin methylation was detected in 92% of cancers arising in the proximal colon (cecum, ascending and transverse colon), 67% of cancers arising in the distal colon (descending and sigmoid colon), and in 80% of cancers of the rectum (Table 2, group A).
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Sensitivity of Detecting Aberrant Vimentin Methylation
To evaluate the potential use of increased vimentin gene methylation as a cancer biomarker, we tested the technical limits to the sensitivity of detecting DNA methylation by primer set 29. This primer set robustly detected vimentin methylation in colon cancer cell lines but not in normal colonic mucosa obtained from control noncancer colon resections (Fig. 3, D). Indeed, DNA from normal colonic mucosa remained negative in this assay, even after subjecting an aliquot of the MS-PCR to a second round of PCR amplification (i.e., 90 cycles total) (Fig. 3, D). Moreover, when DNA from a methylated colon cancer cell line was diluted into DNA from normal colon mucosa, the MS-PCR could detect as little as 2550 pg of input methylated DNA, even in the presence of a 500- to 1000-fold excess of control normal mucosal DNA (Fig. 3, E). This amount of DNA corresponds to a detection limit for the assay of approximately 15 methylated cells.
Detection of Vimentin Methylation in Fecal DNA of Colon Cancer Patients
We next evaluated the ability of MS-PCR primer set 29 to perform as a diagnostic marker for detection of colon cancer by testing its ability to detect aberrant vimentin exon 1 methylation in fecal DNA samples prepared from the stools of 94 additional colorectal cancer patients. Fecal DNAs from 43 of these 94 patients tested positive for vimentin methylation in this assay, yielding a 46% clinical sensitivity for detecting the presence of a colon cancer (95% CI = 36% to 56%) (Table 3). To evaluate the clinical specificity of this assay, we next analyzed fecal DNA from stool samples of 198 control individuals, all of whom were negative for colon cancer on colonoscopic exam. Only 20 samples (10%) tested positive for methylation of vimentin exon 1, for a clinical specificity for the assay of 90% (95% CI = 85% to 94%). We also examined sensitivity for earlier and later stage cancers. The assay had a 43% sensitivity among case patients with early (14) (i.e., stage I and II) colon cancer (26 of 60 samples tested positive) (95% CI = 31% to 57%) and 50% sensitivity among case patients with later (14) stage III and IV colon cancer (17 of 34 samples tested positive) (95% CI = 32% to 68%) (Table 3).
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DISCUSSION |
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Although screening for colon cancer is recommended for all average-risk adults age 50 years and older (22,23), only a minority of the population has demonstrated acceptance of invasive endoscopic-based screening (24). Noninvasive colon cancer screening by testing feces for the presence of occult blood has in one recent large study shown only a 14% sensitivity (25). Thus, molecular assays of fecal DNA for detection of cancer-specific DNA alterations have been proposed as a new approach for screening and detecting early-stage colon cancer (9,26,27). Our finding of 46% sensitivity with 90% specificity for colon cancer detection by assay of fecal DNA for aberrant vimentin exon 1 methylation compares favorably both with testing for fecal occult blood [14% sensitivity and 95% specificity in one recent study (8,25,2831)] and with previous testing of fecal DNA for other DNA biomarkers tested either individually or in combinations (8,25,2831) [52% sensitivity and 94% specificity for a combination panel of such markers (8,25,2831)]. Thus, assay of fecal DNA using vimentin MS-PCR primer pair 29 may have potential clinical utility for colon cancer detection.
Previous investigators have shown that methylated SFRP2 DNA can be detected in the fecal DNA of colon cancer patients with a sensitivity of 77% and a specificity of 77% (8). The 90% specificity for the assay of vimentin methylation may make this assay particularly well suited for inclusion in panels that test for multiple colon cancerassociated mutation or DNA methylation events, in which the challenge is to preserve assay specificity. We have found that the sensitivity of hybrid capture of individual DNA targets from feces is fully maintained in such panels (A.S., unpublished data). Examples of other colon cancerspecific DNA changes that have been detected in fecal DNA include mutations in APC, K-Ras, p53, BAT-26, and the presence of long amplifiable DNA (26,2832). Recent findings of a large prospective trial demonstrated that assessing fecal DNA with panel of these markers achieved 52% sensitivity and 94% specificity for colon cancer detection (25). The 46% sensitivity and 90% specificity of the single assay of fecal DNA for vimentin gene methylation suggests that the further incorporation of this assay into such a marker panel may provide one route for the advancement of this approach.
Our findings of 46% sensitivity for detection of vimentin methylation in stool compare favorably with the estimate of 53% occurrence of vimentin exon methylation noted in our 107tumor sample validation set and is consistent with our observation that the assay for vimentin methylation is technically highly robust. However, the finding of 10% false-positive assays in fecal DNA is somewhat higher than the 2% false-positive rate that we noted in testing normal colon tissues (Fig. 2, D; Table 1). This may in part reflect the larger sample size of the 198 fecal DNA preparations obtained from normal control individuals compared with the set of 58 normal colon mucosa tissues that we initially examined (Tables 1 and 2). However, it is also possible that the higher rate of false-positive tests in the fecal DNA samples reflects a better representation of the total colonic load of vimentin methylation within aberrant crypt foci by fecal DNA collections than by individual tissue samples.
Because the vimentin gene is not expressed by normal colonic epithelial cells, aberrant vimentin gene methylation at exon 1 would not be expected to alter vimentin gene expression or to confer a selective advantage upon colon cancer cells. Thus, we hypothesize that some structural feature of the vimentin exon 1 region must make this a highly favorable target for aberrant methylation early during colon neoplasia. This finding may thus illustrate the use of this broader class of potentially methylated DNA sequences to be developed as robust biomarkers of different cancers.
The detection of aberrant vimentin exon 1 methylation in human fecal DNA identified nearly half of all colon cancer patients in our study with 90% specificity. Given this initial promise, we will now be using this assay in prospective studies as part of a multicenter trial aimed at defining an optimal biomarker panel for colon cancer detection by molecular testing of fecal DNA.
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NOTES |
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We thank Hui Li for helpful scientific discussion and Kory Thornburg and Pam Shaw for helpful technical assistance. Portions of the technology presented in this article are the subject of commercial discussions between Exact Sciences and Case Western Reserve University. J. Olsen and A. P. Shuber are employees and stockholders of Exact Sciences.
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Manuscript received February 1, 2005; revised May 20, 2005; accepted June 23, 2005.
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