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`
` Detection in Fecal DNA of Colon Cancer – Specifi c
`Methylation of the Nonexpressed Vimentin Gene
` Wei-Dong Chen , Z. James Han , Joel Skoletsky , Jeff Olson , Jerome Sah , Lois
` Myeroff , Petra Platzer , Shilong Lu , Dawn Dawson , Joseph Willis , Theresa P.
` Pretlow , James Lutterbaugh , Lakshmi Kasturi , James K. V. Willson , J. Sunil
` Rao , Anthony Shuber , Sanford D. Markowitz
`
` Background: Increased DNA methylation is an epigenetic
` alteration that is common in human cancers and is often
` associated with transcriptional silencing. Aberrantly methyl-
`ated DNA has also been proposed as a potential tumor marker.
`However, genes such as vimentin, which are transcriptionally
`silent in normal epithelium, have not until now been
` considered as targets for cancer-associated aberrant methyl-
`ation and for use as cancer markers. Methods: We applied
`methylation- specifi c polymerase chain reaction to the vimen-
`tin gene, which is transcriptionally silent in normal colono-
`cytes, and compared methylation of vimentin exon 1 in cancer
`tissues and in fecal DNA from colon cancer patients versus
`control samples from healthy subjects. Results: Vimentin
`exon-1 sequences were unmethylated in 45 of 46 normal
`colon tissues. In contrast, vimentin exon-1 sequences were
`methylated in 83% (38 of 46) and 53% (57 of 107) of tumors
`from two independently collected groups of colon cancer
` patients. When evaluated as a marker for colon cancer detec-
`tion in fecal DNA from another set of colon cancer patients,
`aberrant vimentin methylation was detected in fecal DNA
`from 43 of 94 patients, for a sensitivity of 46% (95% confi -
`dence interval [CI] = 35% to 56%). The sensitivity for detect-
`ing stage I and II cancers was 43% (26 of 60 case patients)
`(95% CI = 31% to 57%). Only 10% (20 of 198 case patients)
`of control fecal DNA samples from cancer-free individuals
`tested positive for vimentin methylation, for a specifi city of
`90% (95% CI = 85% to 94%). Conclusions : Aberrant meth-
`ylation of exon-1 sequences within the nontranscribed vimen-
`tin gene is a novel molecular biomarker of colon cancer and
`can be successfully detected in fecal DNA to identify nearly
`half of individuals with colon cancer. [J Natl Cancer Inst
`2005;97:1124 – 32]
`
` Aberrant (i.e., increased) methylation of CpG-rich sequences
`(CpG islands) is an epigenetic change that is common in human
`cancers ( 1 – 4 ) . Such CpG islands are most frequently located in
`the promoter regions or in untranslated fi rst exons of human
`genes ( 1 – 4 ) . Most commonly, increased CpG methylation of
`gene promoters or fi rst exons is associated with loss of gene
`transcription ( 1 – 4 ) . In human colon cancers, several genes have
`been identifi ed that are commonly unmethylated and expressed
`in normal colon mucosa but are methylated and silenced in co-
`lon cancer ( 1 – 7 ) . There has been substantial interest in attempt-
`ing to adapt such cancer-associated aberrant gene methylation
`for use as a marker for potential early detection of colon and
`other cancers ( 3 , 8 – 12 ) .
` 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 confi ned to the bowel wall, surgical cure rates
`
` Affi liations 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 Epidemi-
`ology 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@cwru.edu ).
` See “ Notes ” following “ References. ”
` DOI: 10.1093/jnci/dji204
` © The Author 2005. Published by Oxford University Press. All rights reserved.
`For Permissions, please e-mail: journals.permissions@oupjournals.org .
`
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`are 90% and 75%, respectively ( 14 ) . In contrast, chances for
`cure drop rapidly once colon tumors have spread beyond the
`confi nes of the bowel. Initial reports have confi rmed the poten-
`tial for early detection of colon cancer – derived aberrantly
`methylated DNA in both patient blood and feces, but the sensi-
`tivity and specifi city of currently identifi ed 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 aber-
`rant methylation event. We chose for this approach the vimentin
`gene, which encodes a protein constituent of intermediate fi la-
`ments and whose expression is considered a classic marker of
`mesenchymal cells, such as fi broblasts ( 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.
`
` M ATERIALS AND M ETHODS
`
` Tissues, Cell Lines, and Nucleic Acid Isolation
`
` 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 sam-
`ples 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 (desig-
`nated 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 trans-
`verse 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 screen-
`ing 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 isothio-
`cyanate and fractionation through cesium chloride as previously
`described ( 17 ) .
`
` Immunohistochemistry
`
` Vimentin protein expression in paraffi n-embedded normal
` colon tissue and colon tumors were evaluated using a mouse
`anti-vimentin monoclonal antibody, V9 (DAKO Cytomation,
`Carpinteria, CA). Briefl y, 5- μ m sections of formalin-fi xed, paraf-
`fi n embedded-tissues were deparaffi nized and rehydrated through
`
`graded alcohols to water. Antigen unmasking was performed by
`heat treatment (10 m M 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, fol-
`lowed by hematoxylin counterstaining. In every analysis, longi-
`tudinally 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 epithe-
`lial 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 350 g for
`5 minutes at 4 °C.
`
` Real-time Reverse Transcription – Polymerase
`Chain Reaction
`
` The vimentin transcript was amplifi ed from the isolated RNA
`of normal colon and colon cancer tissues and colon cancer –
` derived cell lines in an iCycler instrument (BioRad Laborato-
`ries, Hercules, CA) using 400 n M 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- μ L 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 amplifi ed
`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 = 2 CTvimentin – CTMuc2 , where CT vimentin
`is the cycle number for crossing the iCycler detection threshold
`in real-time PCR amplifi cation of vimentin, and CT Muc2 is the
`cycle number for crossing the iCycler detection threshold in real-
`time PCR amplifi cation of Muc2.
`
` Bisulfi te Conversion of Genomic DNA and MS-PCR
`
` Bisulfi te conversion of DNA was performed as described
` previously ( 6 , 18 ) to create a template for methylation-specifi c
`PCR (MS-PCR). Briefl y, 500 ng to 2 μ g of genomic DNA from
`each sample in a volume of 50 μ L was denatured by NaOH
`(freshly made, fi nal concentration, 0.2 M ) at 37 °C for 15 min-
`utes. Next, 30 μ L of 10 m M fresh hydroquinone and 520 μ L of
`freshly prepared 3.0 M NaHSO 3 , pH 5.0 (Sigma, St. Louis, MO)
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`were added, and the mixture was incubated at 55 °C for 16 hours.
` Bisulfi te-modifi ed DNA was purifi ed using the Wizard DNA
`Cleanup kit (Promega, Madison, WI). The DNA was desulfo-
`nated by incubation with NaOH at a fi nal concentration of 0.3 M
`at room temperature for 15 min and neutralized by adding
` ammonium acetate, pH 7.0, to a fi nal concentration of 3 M . DNA
`was precipitated with ethanol and resuspended in distilled
`water to a fi nal concentration of 5 ng/ μ L.
` Bisulfi te-treated DNA was then used as the template for
`MS-PCR, which was performed as described previously ( 6 , 18 ) .
`Briefl y, 5 µL of bisulfi te-converted genomic DNA served as the
`PCR template. The amplifi cation was in a reaction of 25 μL
` containing 0.19 m M each dNTP, 1.5 m M MgCl2, 400 n M of
` forward and reverse primers, and 1.25 U of AmpliTaq Gold in the
`recommended buffer. Amplifi cation primers and reaction con-
`ditions are provided (Supplementary Table, available at http://
`jncicancerspectrum.oxfordjournals.org/jnci/content/vol97issue15 ).
`Vimentin MS-PCR reaction #29 employed forward amplifi cation
`primer 5 ′ -TCGTTTCGAGGTTTTCGCGTTAGAGAC-3 ′ and
`reverse amplifi cation 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 amplifi cations from fecal
`DNA, both forward and reverse MS-PCR primers were addition-
`ally 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 amplifi cation of templates that had incorporated the PCR
`primers. For sequencing of bisulfi te-converted DNA, products
`were amplifi ed with methylation-indifferent primers and cloned
`into pCR2.1 TOPO TA cloning vector (Invitrogen, Carlsbad,
`CA); 10 – 15 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 repre-
`sented 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). Ho-
`mogenized samples were then archived at − 80 °C for an average
`of 12 months (range = 6 – 18 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 superna-
`tants were treated with 20 μ L 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 1× TE buffer (0.01 M Tris, pH 7.4, 0.001 M EDTA)
`(Pierce, Rockford, IL). Target human vimentin DNA fragments
`were purifi ed from total DNA preparations by acrylamide gel –
` based affi nity capture as previously described ( 19 ) . Total DNA
`yields from normal patients (median = 936 genome equivalents,
`range = 33 – 18 560 genome equivalents) and cancer patients
` (median = 1014 genome equivalents, range = 32 – 3700 genome
`equivalents) were similar. Total captured DNA from each sample
`was then subjected to bisulfi te-modifi cation and MS-PCR, and
`the results were analyzed in a manner blinded to patient’s disease
`status.
`
` Statistical Analysis
`
` Exact 95% confi dence 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 signifi cant
`if P <.05. MS-PCR reactions were run independently in qua-
`druplicate 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.
`
` R ESULTS
`
` Expression and Methylation of the Vimentin Gene
`in Colon Cancer Cell Lines, Colon Cancers, and
`Normal Colonocytes
`
` 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 can-
`cers ( Fig. 1 , A). To confi rm that the vimentin gene is transcrip-
`tionally 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 purifi ed 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 non-
`epithelial cell population.
` The structure of the vimentin gene demonstrates a dense re-
`gion of CpG dinucleotides starting upstream of the fi rst exon and
`continuing across the fi rst two-thirds of this exon ( Fig. 2, A ). To
`assay this region for potential cytosine methylation, we designed
`a series of eight MS-PCR primer pairs that defi ned overlapping
`fragments spanning the region. These primers were initially used
`to assay the nonexpressed vimentin gene for DNA methylation in
`normal colonic mucosal samples from 12 control individuals
`who did not have colon cancer and in 11 colon cancer cell lines.
`Although the vimentin gene is transcriptionally silent in colonic
`mucosal epithelial cells, a 5 ′ portion of vimentin exon 1, defi ned
`by six overlapping MS-PCR primers, was free of detectable
`DNA methylation ( Fig. 2 , B and Table 1 ) in either 11 or 12 of
`
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` Fig. 1. Localization of vimentin expression. A ) Immunohistochemical detection
`of vimentin expression ( brown ) using the monoclonal V9 anti-vimentin
`antibody (DAKO Cytomation, Carpinteria, CA) in tissue sections from two
`normal colonic mucosa specimens and two colon cancers. Comparison with
`a hematoxylin counterstain shows clear vimentin staining in stromal cells
`and lymphocytes but not in epithelial cells. Bar = 100 μ m. B ) Phase-contrast
`microscopy of colonic crypt preparations showing the enrichment of epithelial
`cells compared with the colonic mucosa specimens. Bar = 100 μ m. C ) Levels
`of vimentin transcript expression measured by real-time PCR in colonic crypt
`
`preparations ( solid bars , labeled C) and in matched colonic mucosal tissue
`samples ( open bars , labeled M) for three different individuals (case patients
`476, 505, and 1026). For each patient, levels of vimentin expression in the
`mucosal tissue were set to equal 100%. Vimentin expression in mucosa and
`in crypt preparations was normalized to the epithelial cell mass of the tissue
`as assessed by the level of expression of the colonic epithelial cell marker
`Muc2. Graphed values are means and 95% confi dence intervals of triplicate
`determinations. Where 95% confi dence intervals are not visible, they represent
`a range of less than 1%.
`
`the 12 normal colonic mucosa samples assayed. In contrast, the
`11 colon cancer cell lines demonstrated clear acquisition of aber-
`rant methylation across vimentin exon 1, with different MS-PCR
`primer pairs detecting methylation in from eight to 10 of the
`11 cell lines assayed ( Fig. 2 , C and Table 1 ).
` To quantify the extent of vimentin exon 1 methylation in the
`11 cancer cell lines, we prepared bisulfi te-converted DNA from
`three of these cell lines (Vaco5, Vaco6, Vaco400). For each cell
`line, we sequenced vimentin exon 1 from multiple individual
`PCR-amplifi ed clones and assessed whether the antecedent cyto-
`sine at each CpG site was methylated or unmethylated. In this
`analysis, every CpG cytosine within the target vimentin exon 1
`sequence was methylated in every clone sequenced, demonstrat-
`ing that in all of these cell lines this region had become essen-
`tially 100% methylated (data not shown).
`
` 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 defi ned 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 meth-
`ylation 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, ascend-
`ing 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).
` To confi rm these results, we used primer set 29 to assay a
`second independent collection of 107 colon cancer samples.
`Again, a majority, 53% (57 of 107 case patients), of this second
`set of colon cancer case patients demonstrated aberrant vimentin
`methylation. In this second patient series, 72% of cancers of the
`proximal colon assayed positive for aberrant vimentin gene
`methylation, and 45% of cancers of the distal colon were meth-
`ylated ( Table 2 , group B). The smaller proportion of proximal
` colon cancers in the second patient cohort than the fi rst likely
`accounts for this series having a somewhat lower overall
` frequency of vimentin methylation ( Table 2 ). Also, in both
` series of tumors, early stage I and stage II cancers that have not
`spread beyond the wall of the colon showed rates of vimentin
`gene methylation at least equal to those of later stage III and
`stage IV cancers ( Table 2 ). Detection of vimentin gene methyla-
`tion was technically robust, and all normal samples that tested
`negative for vimentin gene methylation tested positive for a
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` Fig. 2. Distribution of cytosine methylation across the
`vimentin gene. A ) Balloons designate the distribution of
`CpG dinucleotides across the promoter and fi rst exon of
`the vimentin gene. B ) Different vimentin gene domains that
`were amplifi ed by a panel of methylation specifi c polymerase
`chain reactions (MS-PCRs) that were applied to normal colon
`mucosa samples from 12 control noncancer patients (Normal
`Group 1). C ) Using the same convention as in panel B, the
`results of MS-PCR assays for vimentin gene methylation in
`11 colon cancer cell lines are shown. D ) The results of MS-
`PCR testing for vimentin gene methylation in normal colonic
`mucosa samples from 46 colon cancer patients. E ) The results
`of MS-PCR testing for vimentin gene methylation in colon
`tumor samples from the same 46 patients represented in
`panel D. The color code of the line denoting each MS-PCR
`indicates the percentage of case patients testing positive for
`methylation ( green = <10% of samples methylated, yellow =
`10% – 49%, pink = 50% – 80%, and red = >80%.
`
`A
`
`B
`
`Normal
`Group-1
`
`C
`
`Cancer
`Cell
`Lines
`
`D
`
`Normal
`Group-2
`
`E
`
`Cancer
`Tissues
`
`ATG
`
`57800
`
`57900
`
`58000
`
`58100
`
`exon 1
`58200
`
`58300
`
`58400
`
`58500
`
`58600
`
`58700
`
`MSP2
`
`MSP2
`
`MSP3
`MSP28
`MSP29
`
`MSP3
`MSP28
`MSP29
`
`MSP3
`MSP29
`
`MSP3
`MSP29
`
`MSP36
`MSP37
`MSP41
`MSP50
`
`MSP36
`
`MSP37
`MSP41
`MSP50
`
`MSP36
`MSP37
`MSP41
`MSP50
`
`MSP36
`MSP37
`MSP41
`MSP50
`
` constitutively methylated control (MS-PCR assay F1-19M)
`( Fig. 3, A ).
` To determine the timing during colon carcinogenesis of
` ac quisition of vimentin gene methylation, we next used MS-
`PCR primer set 29 to test a set of 10 colonic adenomas of 1 cm or
`greater in size. Of these 10 adenoma lesions, seven were positive
`for vimentin gene methylation ( Fig. 3, B ). We therefore used
`MS-PCR primer set 29 to assay DNA extracted from aberrant
`crypt foci, the microscopic lesions that are recognized as the
` earliest morphologic abnormality of the colonic mucosa ( 21 ) . Of
`nine aberrant crypt foci obtained from colons of six different
` individuals, seven aberrant crypts from fi ve different individuals
`were positive for vimentin gene methylation ( Fig. 3, C ). In con-
`trast, only one of 14 microdissected regions of histologically
`
` normal colon from these individuals tested positive for aberrant
`vimentin methylation ( Fig. 3, C ).
`
` Sensitivity of Detecting Aberrant Vimentin Methylation
`
` To evaluate the potential use of increased vimentin gene meth-
`ylation 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 can-
`cer 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 amplifi cation (i.e., 90 cycles total) ( Fig. 3, D ). Moreover,
`
` Table 1. Vimentin exon-1 methylation in normal and cancer tissues and cancer cell lines *
`
` Primer set
`
` Normal group 1 † (n = 12)
`
` Cancer cell lines (n = 11)
`
` Normal group 2 ‡ (n = 46)
`
` Cancer tissues (n = 46)
`
` MSP2
` MSP3
` MSP28
` MSP29
` MSP36
` MSP37
` MSP41
` MSP50
`
` 83 (52 to 98)
` 0 (0 to 22)
` 83 (52 to 98)
` 0 (0 to 22)
` 8 (0 to 39)
` 0 (0 to 22)
` 0 (0 to 22)
` 8 (0 to 39)
`
` 91 (59 to 99)
` 82 (48 to 98)
` 91 (59 to 99)
` 82 (48 to 98)
` 82 (48 to 98)
` 82 (48 to 98)
` 73 (39 to 94)
` 82 (48 to 98)
`
` —
` 0 (0 to 6)
` —
` 2 (01 to 12)
` 22 (11 to 36)
` 24 (13 to 39)
` 46 (21 to 61)
` 24 (13 to 39)
`
` —
` 63 (48 to 77)
` —
` 83 (69 to 92)
` 87 (74 to 95)
` 89 (76 to 96)
` 89 (76 to 96)
` 87 (74 to 95)
`
` * The percentage of subjects demonstrating vimentin gene methylation from methylation-specifi c polymerase chain reaction assays using the primer sets shown.
` † Normal group 1 = normal colon mucosal tissues from non – colon cancer patients.
` ‡ Normal group 2 = matched normal colonic mucosa from colon cancer patients whose cancer tissues were assayed; — = Normal group 2 and cancer tissues groups
`were not assayed with primer sets 2 and 28.
`
`1128 ARTICLES
`
`Journal of the National Cancer Institute, Vol. 97, No. 15, August 3, 2005
`
`Geneoscopy Exhibit 1057, Page 5
`
`

`

`Downloaded from https://academic.oup.com/jnci/article/97/15/1124/2521303 by guest on 30 December 2023
`
` Table 2. Detection of aberrant vimentin exon-1 methylation in colon cancer tissue, according to tumor location and tumor stage *
`
`
` Tumor characteristic
`
` Location
`
` Proximal colon
`
` Distal colon
`
` Rectum
`
` Unknown
` Stage
`
` I
`
` II
`
` III
`
` IV
`
` Unknown
`
`
` Total
`
` Sample number
`
` Group A
` No. positive
`
` Positive, %
`
` Sample number
`
` Group B
` No. positive
`
` Positive, %
`
`
` 26
` 15
` 5
` 0
`
` 0
` 18
` 15
` 13
` 0
` 46
`
`
` 24
` 10
` 4
` 0
`
` 0
` 17
` 11
` 10
` 0
` 38
`
`
` 92
` 67
` 80
` 0
`
` 0
` 94
` 73
` 77
` 0
` 83
`
`
` 29
` 29
` 21
` 28
`
` 2
` 43
` 26
` 17
` 19
` 107
`
`
` 21
` 13
` 8
` 15
`
` 1
` 25
` 15
` 7
` 9
` 57
`
`
` 72
` 45
` 38
` 54
`
` 50
` 58
` 58
` 41
` 47
` 53
`
` * Results are shown for two independent sets of colon cancers, group A (with 46 case patients) and group B (with 107 case patients).
`
`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 25 – 50 pg of input methylated DNA, even in the pres-
`ence 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 specifi city 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 sam-
`ples (10%) tested positive for methylation of vimentin exon 1,
`for a clinical specifi city 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 sam-
`ples tested positive) (95% CI = 31% to 57%) and 50% sensitiv-
`ity 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 ).
` Moreover, detection of vimentin exon 1 methylation in fecal
`DNA with the primer set 29 assay was equally sensitive for
` detecting colon cancers arising proximal to the splenic fl exure
`(46% sensitivity) and those arising distal to the splenic fl exure
`(45% sensitivity) ( Table 3 ). Colon cancer patients with positive
`fecal DNA tests were not statistically signifi cantly different from
`those with negative tests with respect to age or sex ( Table 4 ).
`Similarly, among noncancer control subjects, individuals with
`false-positive fecal DNA tests were not statistically signifi cantly
`different from those with negative tests with respect to age, sex,
`or history of colon cancer in a fi rst-degree relative ( Table 4 ).
`False-positive fecal DNA tests were somewhat more common
`among control patients with hyperplastic or adenomatous polyps
`
`than among those with no evidence of polyps, but these differ-
`ences were not statistically signifi cant ( Table 4 ).
` The fecal DNA samples used in this study were derived from
`four different medical care organizations, of which one group
`contributed half of the total samples studied and the remaining
`three groups contributed the balance of the samples. However,
`the sensitivity for detection of colon cancer by assay of vimentin
`gene methylation in fecal DNA was essentially identical for case
`patients from the largest donor site as for the case patients
` contributed by the remaining three sites combined (data not
`shown). Thus, our fi ndings appear to be reproducible in at least
`two different patient cohorts.
`
` D ISCUSSION
`
` This study has defi ned a DNA sequence within vimentin exon 1
`that is commonly targeted for aberrant DNA methylation by hu-
`man colon cancers, particularly those arising in the prox