Comparative Analysis of PCR-Based
Biomarker Assay Methods for Colorectal
Polyp Detection from Fecal DNA
Christoph Ausch,1,2†Young-Ho Kim,1,3†Karen D.
Tsuchiya, Slavomir Dzieciatkowski,1Mary K. Wash-
ington,4Cristos Paraskeva,5Jerry Radich,1and
William M. Grady1*
1Division of Clinical Research, Fred Hutchinson Can-
cer Research Center, Seattle, WA, USA;2Institute for
Surgical Oncology, Cluster for Translational Oncol-
ogy, Ludwig Boltzmann Research Institute, Danube
Hospital, Vienna, Austria;3Department of Medicine,
Samsung Medical Center, Sungkyunkwan University
thology, Vanderbilt University Medical School, Nash-
ville, TN, USA;5University of Bristol, Bristol, UK;
* address correspondence to this author at: Fred
Hutchinson Cancer Research Center, 1100 Fairview
e-mail firstname.lastname@example.org.†These authors contributed
equally to this study.
BACKGROUND: Aberrantly methylated genes are prom-
ising biomarkers for the detection of colon adenomas
and colorectal cancers (CRCs). The optimal assay type
and specific methylated genes for these assays remain
to be determined.
METHODS: We used genomewide microarray-based as-
says to identify methylated genes as candidate biomar-
kers for colon neoplasms. The frequency of aberrant
methylation of these genes in primary tumors was as-
its of detection and specificities for different types of
PCR-based assays were then assessed with the most
promising genes identified in this screen. Finally, we
assessed the best-performing MSP assay as an early-
detection marker for fecal DNA samples.
pha 4 subunit of VLA-4 receptor)] was identified as a
novel gene frequently methylated in CRC. Methylated
ITGA4 is present in 75% of colon adenomas (n ? 36)
and 92% of colon adenocarcinomas (n ? 75). Com-
parison of end point MSP, end point MSP with
clamped primers, and quantitative fluorescent MSP
(qMSP) approaches revealed that both types of end
point MSP assays could routinely detect as little as 70
pg DNA, whereas the qMSP assay could routinely de-
ylated ITGA4 can detect 69% of individuals with colon
(n ? 28).
CONCLUSIONS: Methylated ITGA4 is a promising
marker gene for early detection of colonic neoplasms.
types tested, and a qMSP assay that detects methylated
ITGA4 has potential as an early-detection assay for co-
Mortality related to colorectal cancer (CRC)6can be
reduced by screening with fecal occult blood tests and
is optimal, however, because of low positive predictive
values and low compliance, respectively. Thus, a sub-
stantial effort has been made to develop molecular
marker assays that function as diagnostically accurate
noninvasive markers for the early detection of colon
adenomas and cancers and that have the potential to
increase compliance. The use of stool-based DNA
tests are more diagnostically specific than fecal occult
the currently commercially available assay for detect-
ing colon adenomas appears to be ?50% (4, 5). Some
of this lack of diagnostic sensitivity appears to be re-
lated to the particular molecular markers assessed
(6, 7). Another reason for the suboptimal diagnostic
sensitivity may be secondary to technical aspects re-
lated to the assay design (8). Thus, we performed a
genomewide screen for novel methylated genes that
pared the performance characteristics of 3 different
types of PCR-based assays for detecting these methyl-
For the studies described below, we used the
VACO 235 and AA/C1 colorectal adenoma cell lines
and the VACO 411, AA/C1/SB10, HT29, RKO, and
SW48 CRC cell lines. The cell lines were grown as pre-
to extract total RNA to be used in the microarray anal-
and carried out a second purification step with the
performed according to the manufacturers’ protocols.
We used both gene-unmasking methods and methods
based on methylated CpG island amplification mi-
croarrays to identify genes that are aberrantly methyl-
ated in colon neoplasms (11, 12). Microarray data
were analyzed with the aid of GenePix Pro (Molecular
6Nonstandard abbreviations: CRC, colorectal cancer; MSP, methylation-specific
PCR; qMSP, quantitative fluorescent MSP.
Clinical Chemistry 55:8
http://www.clinchem.org/cgi/doi/10.1373/clinchem.2008.122937 The latest version is at
Papers in Press. Published June 18, 2009 as doi:10.1373/clinchem.2008.122937
Copyright (C) 2009 by The American Association for Clinical Chemistry
in the Data Supplement that accompanies the online
version of this Brief Communication at http://www.
clinchem.org/content/vol55/issue8. These studies identi-
ITGA47[integrin, alpha 4 (antigen CD49D, alpha 4 sub-
ylated in primary colon neoplasms. Methylated ITGA4
and in CRC cell lines AA/C1/SB10, VACO 411, HT29,
RKO, and SW48 (see Fig. 1 in the online Data Supple-
ment). To confirm methylation of the CpG island in the
ITGA4 promoter, we performed bisulfite genomic se-
quencing of ITGA4 in cell lines AA/C1, VACO 235, AA/
C1/SB10, and VACO 411 (for methods see File 1 in the
online Data Supplement). The CpG island of exon 1 was
the methylation-specific PCR (MSP) assays for methyl-
methylation status determined by bisulfite sequencing
(see Fig. 2 in the online Data Supplement). Quantitative
reverse-transcription PCR was performed as previously
substantially increased after treatment of AAC1/SB10
with 5-aza-2?-deoxycytidine, a result consistent with
Data Supplement describes the conditions for MSP and
of ITGA4 in samples of nonpathologic colon mucosa
(n ? 32) and colon neoplasms. Colorectal tumor sam-
lected from patients treated at Vanderbilt University
Medical Center, the VA Tennessee Valley Health Care
System, Meharry Medical Center (Nashville, TN), and
Samsung Medical Center (Seoul, Korea). Informed
cordance with protocols approved by the institutional
review board of each institution (see Table 2 in the
In addition, 32 nonpathologic colon samples resected
for benign disease were obtained from the Vanderbilt
was 62 years (range, 35–80 years) (14). DNA was ex-
tracted and analyzed by MSP as previously described
7Human genes: ITGA4, integrin, alpha 4 (antigen CD49D, alpha 4 subunit of
VLA-4 receptor); CDKN2A, cyclin-dependent kinase inhibitor 2A (melanoma,
p16, inhibits CDK4); MGMT, O-6-methylguanine-DNA methyltransferase; MLH1,
mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli).
Fig. 1. The qMSP assay for methylated ITGA4.
(A), Example results for the methylated ITGA4 qMSP assay with serially diluted DNA, demonstrating both consistent replicate
results and an increasing threshold cycle number for samples with progressively less input DNA. The absolute amounts of DNA
used for each replicate are shown. The DNA source was CRC cell line SW48. (B), Reproducibility of the methylated ITGA4 qMSP
assay with serial dilutions of DNA from the SW48 cell line. Black dots represent results from a qMSP assay run with a sample
with the indicated amount of input DNA; gray dot represents a result that was ?1 SD from the mean value.
Clinical Chemistry 55:8 (2009)
(15). Methylated ITGA4 was detected in 6% of the
nonpathologic samples of colon mucosa (n ? 32), in
75% of adenomas (n ? 27), and in 92% of CRCs (n ?
69). Methylated ITGA4 was more common in cases of
advanced adenoma than in early adenoma [87% (n ?
23) vs 54% (n ? 13); P ? 0.046, Fisher exact test].
We then evaluated the analytical performance
characteristics of 3 different types of PCR-based assays
[cyclin-dependent kinase inhibitor 2A (melanoma,
p16, inhibits CDK4)], MGMT (O-6-methylguanine-
lon cancer, nonpolyposis type 2 (E. coli)], and ITGA4.
We compared end point MSP, end point MSP with
clamped PCR primers, and quantitative fluorescent
MSP (qMSP) to determine which assay type had the
best limit of detection (16, 17). We conducted this
comparison with methylated and unmethylated DNA.
For methylated DNA, we extracted genomic DNA
CDKN2A, MGMT, MLH1, and ITGA4 and treated it
with the CpG DNA methylase SssI (methylated DNA
control). For unmethylated DNA, we used genomic
DNA from peripheral blood leukocytes, which carry
the unmethylated CDKN2A, MGMT, MLH1, and
ITGA4 genes (unmethylated DNA control). Initially,
we performed a dilution experiment to determine the
technical detection limits of the assay types. We ana-
lyzed serial dilutions of bisulfite-treated DNA from
MLH1, and ITGA4. The end point MSP assay with
clamped primers had a 10-fold lower detection limit
for methylated CDKN2A and a 2.5-fold lower detec-
tion limit for MLH1 and could detect as little as 7 pg of
modified primer; however, the detection limit for
methylated ITGA4 was 70 pg with either the un-
clamped or clamped primer (see Table 3 in the online
of studies with qMSP assays to determine if this assay
type would affect the technical sensitivity of the assay
for methylated ITGA4. Application of the qMSP as-
say to the same serially diluted samples revealed a 10-
fold improvement for detecting methylated ITGA4,
(Fig. 1A). Thus, the effect of the different assay meth-
ods appears to vary with the gene; the assay with the
We next tested the potential of the qMSP assay to
detect methylated ITGA4 in samples of human fecal
DNA. The fecal DNA samples from patients with his-
tologically confirmed polyps (n ? 13) and patients
Table 1. ITGA4 and Alu-C4 qMSP results from fecal
Tubular adenomas 23.2
aThe Alu-C4 assay was used to assess the total human DNA present in the
fecal DNA sample. The assay was run as previously described [Weisen-
berger et al. (18)].
bThe cutoff threshold cycle (CT) value for methylated ITGA4 is 38.
cM, ; NA, no amplification; ND, not determined; —, not present; U, ●●●.
Clinical Chemistry 55:8 (2009)
(n ? 28) were obtained as previously described (14).
The sample-collection method is described in File 1 in
the online Data Supplement. The mean r2value of the
calibration curves performed in 7 experiments was
0.988 (range, 0.98–0.99; Fig. 1B). Methylated ITGA4
was found in 69% (9 of 13) of the patients with colon
adenomas and in 21% (6 of 28) of people with no
polyps detected by colonoscopy (Table 1). We also as-
polyps carrying methylated ITGA4, as detected by the
end point MSP assay. We found that the fecal DNA–
mative cases) of the patients with polyps that carried
plement). The fecal DNA–based assay had a lower di-
agnostic sensitivity and specificity than the tissue-
based results. We believe this finding reflects the facts
that the fecal samples were collected without the use of
a stabilization buffer and that capture probes were not
used to enrich for methylated ITGA4, which are tech-
nical limitations that will be addressed in future stud-
the samples is from the nonpathologic colon epithe-
lium and therefore not relevant for assessing the
(which is an indicator of the total amount of input
all amount of DNA in the samples (see File 1 in the
measure Alu DNA).
DNA–based early-detection marker for CRC. The
ylated ITGA4. The ITGA4 qMSP assay that we have
designed can detect 1 genome equivalent of tumor
DNA and has a 73% diagnostic sensitivity for patients
with colon adenomas when the assay is used on fecal
MSP is the most diagnostically sensitive assay type;
however, qMSP is usually desirable because optimal
thresholds can be determined to maximize the perfor-
mance of the assay for specific clinical applications.
Further assessment of methylated ITGA4 as an early-
detection marker and development of additional
qMSP-based assays for other genes methylated in CRC
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon
manuscript submission, all authors completed the Disclosures of Poten-
tial Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: J. Radich, Novartis, Bristol-Myers
Stock Ownership: None declared.
Honoraria: J. Radich, Novartis, Bristol-Myers Squibb.
Research Funding: J. Radich, Novartis, Bristol-Myers Squibb. This
work was supported by funding provided by a Damon Runyon Lilly
Clinical Investigator Award, a Presidential Early Career Award for
Scientists and Engineers (Department of Veterans Affairs), and a
Burroughs Wellcome Fund Clinical Scientist in Translational Re-
search Award (W.M. Grady), and by an Austrian Society of Surgery
Research Grant and a Felix Mandl Research Grant, City of Vienna,
Austria (C. Ausch).
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the
of data, or preparation or approval of manuscript.
Acknowledgment: We thank James K.V. Willson for providing the
VACO 411 and VACO 235 cell lines.
1. Levin B, Lieberman DA, McFarland B, Andrews
KS, Brooks D, Bond J, et al. Screening and sur-
veillance for the early detection of colorectal can-
cer and adenomatous polyps, 2008: a joint guide-
line from the American Cancer Society, the US
Multi-Society Task Force on Colorectal Cancer,
and the American College of Radiology. Gastro-
2. Imperiale TF, Ransohoff DF, Itzkowitz SH, Turn-
bull BA, Ross ME. Fecal DNA versus fecal occult
blood for colorectal-cancer screening in an
average-risk population. N Engl J Med 2004;351:
3. Osborn NK, Ahlquist DA. Stool screening for colo-
rectal cancer: molecular approaches. Gastroenter-
4. Itzkowitz SH, Jandorf L, Brand R, Rabeneck L,
Schroy PC 3rd, Sontag S, et al. Improved fecal
DNA test for colorectal cancer screening. Clin
Gastroenterol Hepatol 2007;5:111–7.
5. Itzkowitz S, Brand R, Jandorf L, Durkee K, Mill-
holland J, Rabeneck L, et al. A simplified, nonin-
vasive stool DNA test for colorectal cancer detec-
tion. Am J Gastroenterol 2008;103:2862–70.
6. Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J,
Myeroff L, et al. Detection in fecal DNA of colon
cancer-specific methylation of the nonexpressed
vimentin gene. J Natl Cancer Inst 2005;97:1124–
7. Zou H, Harrington JJ, Shire AM, Rego RL, Wang L,
Campbell ME, et al. Highly methylated genes in
colorectal neoplasia: implications for screening.
Cancer Epidemiol Biomarkers Prev 2007;16:
8. Diehl F, Schmidt K, Durkee KH, Moore KJ, Good-
man SN, Shuber AP, et al. Analysis of mutations
in DNA isolated from plasma and stool of colo-
rectal cancer patients. Gastroenterology 2008;
9. Willson J, Bittner G, Oberley T, Meisner G, Weese
J. Cell culture of human colon adenomas and
carcinomas. Cancer Res 1987;47:2704–13.
10. Williams AC, Harper SJ, Paraskeva C. Neoplas-
tic transformation of a human colonic epithelial
cell line: in vitro evidence for the adenoma to
carcinoma sequence. Cancer Res 1990;50:
11. Fraga MF, Herranz M, Espada J, Ballestar E, Paz
MF, Ropero S, et al. A mouse skin multistage
carcinogenesis model reflects the aberrant DNA
methylation patterns of human tumors. Cancer
12. Shen L, Kondo Y, Guo Y, Zhang J, Zhang L,
Ahmed S, et al. Genome-wide profiling of DNA
Clinical Chemistry 55:8 (2009)
methylation reveals a class of normally methyl-
ated CpG island promoters. PLoS Genet 2007;3:
13. Rojas A, Meherem S, Kim YH, Washington MK,
Willis JE, Markowitz SD, Grady WM. The aberrant
methylation of TSP1 suppresses TGF-beta1 acti-
vation in colorectal cancer. Int J Cancer 2008;
14. Petko Z, Ghiassi M, Shuber A, Gorham J, Smalley
W, Washington MK, et al. Aberrantly methylated
CDKN2A, MGMT, and MLH1 in colon polyps and
in fecal DNA from patients with colorectal polyps.
Clin Cancer Res 2005;11:1203–9.
15. Grady WM, Willis J, Guilford PJ, Dunbier AK, Toro
TT, Lynch H, et al. Methylation of the CDH1
promoter as the second genetic hit in hereditary
diffuse gastric cancer. Nat Genet 2000;26:16–7.
16. Petkov PM, Zavadil J, Goetz D, Chu T, Carver R,
Rogler CE, et al. Gene expression pattern in he-
patic stem/progenitor cells during rat fetal devel-
opment using complementary DNA microarrays.
17. Eads CA, Lord RV, Wickramasinghe K, Long TI,
Kurumboor SK, Bernstein L, et al. Epigenetic pat-
terns in the progression of esophageal adenocar-
cinoma. Cancer Res 2001;61:3410–8.
18. Weisenberger DJ, Campan M, Long TI, Kim M,
Woods C, Fiala E, et al. Analysis of repetitive
element DNA methylation by MethyLight. Nucleic
Acids Res 2005;33:6823–36.
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