Genome-wide DNA Methylation
Profiling of Cell-Free Serum DNA
in Esophageal Adenocarcinoma
and Barrett Esophagus1,2
Rihong Zhai*, Yang Zhao*, Li Su*, Lauren Cassidy*,
Geoffrey Liu†and David C. Christiani*,‡
*Environmental and Occupational Medicine and
Epidemiology Program, Department of Environmental
Health, Harvard School of Public Health, Boston, MA, USA;
†Medical Oncology and Haematology, Department of
Medicine, Princess Margaret Hospital/Ontario Cancer
Institute, University of Toronto, Ontario, Canada;
‡Department of Medicine, Massachusetts General Hospital,
Boston, MA, USA
Aberrant DNA methylation (DNAm) is a feature of most types of cancers. Genome-wide DNAm profiling has been
performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor
tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm) profiles
reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with
cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative signif-
icance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA) and Barrett esophagus (BE, EA pre-
cursor). We performed genome-wide DNAm profiling in EA tissue DNA (n = 8) and matched serum DNA (n = 8), in
serum DNA of BE (n = 10), and in healthy controls (n = 10) using the Infinium HumanMethylation27 BeadChip that
covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in
matched tumor tissue DNA (r = 0.92) in patients with EA. We selected the most differentially methylated loci to
perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control
samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These
results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in
corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening
of EA and EA premalignant lesions.
Neoplasia (2012) 14, 29–33
DNA methylation (DNAm) is a fundamental epigenetic modifica-
tion in which a methyl group is added to the carbon-5 position of
the cytosine ring within the CpG dinucleotide . Extensive research
has revealed that DNAm is widely implicated in all crucial changes in
cancer cells, such as tumor-suppressor gene silencing, oncogene acti-
vation, and defective DNA repair [2,3]. Indeed, aberrant DNAm has
been detected in a variety of cancers, including esophagus, colon,
breast, liver, kidney, and lung [4–10]. To date, however, most stud-
ies of cancer DNAm have investigated DNAm in tissue-extracted
DNA. The invasive procedure and the likely existence of tissue het-
erogeneity limit the utility of tissue DNA for epidemiologic studies.
Therefore, it is desirable to develop less invasive and more accessible
Abbreviations: EA, esophageal adenocarcinoma; DNAm, DNA methylation; cfDNA,
cell-free circulating DNA
Address all correspondence to: David C. Christiani, MD, MPH, MS, or Rihong Zhai,
MD, PhD, Department of Environmental Health, Harvard School of Public Health,
665 Huntington Ave, FXB109, Boston, MA 02115. E-mail: email@example.com,
1This study was funded by National Institutes of Health grant RO1CA109193,
RO3CA110822, RO1CA074386, P50CA090578, and ES00002 and by the Flight
Attendant Medical Research Institute Award YCSA-062459. All authors disclosed
no conflict of interest; the institutes that provided the grant supports did not partic-
ipate in the study design, the collection, analysis, or interpretation of data.
2This article refers to supplementary materials, which is designated by Figure W1 and
is available online at www.neoplasia.com.
Received 21 November 2011; Revised 10 January 2012; Accepted 11 January 2012
Copyright © 2012 Neoplasia Press, Inc. All rights reserved 1522-8002/12/$25.00
Volume 14 Number 1January 2012pp. 29–33
approaches that can substitute or complement tissue DNA for
The presence of cell-free DNA (cfDNA) in the plasma/serum in
healthy individuals and, in higher amounts, in cancer patients was
demonstrated three decades ago [11,12]. However, it is only recently
possible to use cfDNA as a marker for cancer diagnosis or progression
[13,14]. In cancer patients, cfDNA carries the same mutations (K-ras,
N-ras, p53) as those found in corresponding tumor tissues [15,16].
CfDNA also carries other features of the primary tumor, including
microsatellite instabilities, loss of heterozygosity, and epigenetic
[13,17,18]. Interestingly, DNAm patterns detected in cfDNA are
in high concordance with patterns observed in corresponding primary
tumor tissues [19,20]. Indeed, individual cfDNA methylation
(cfDNAm) markers have been linked to different types of cancer, in-
cluding esophageal adenocarcinoma (EA) [21–27]. However, there
has been relatively little attention on the association of cfDNAm with
cancer precursor; no studies have examined cfDNAm profiles in rela-
tion to cancer precursor and cancers on a genome-wide scale. The
objective of this study was to compare the concordance of genome-
wide DNAm profiles between tumor tissues and sera and to assess the
performance of genome-wide cfDNAm profiles in differentiating EA,
EA precursor (Barrett esophagus, BE), and control.
Materials and Methods
Genomic DNA Extraction from Tissue and Serum
Matched serum and tissue samples were obtained from eight ran-
domly selected EA patients. Additional serum samples were collected
from 10 BE patients and 10 healthy controls [28,29]. EA and BE were
incident cases of histologically confirmed patients, and all subjects were
recruited from Massachusetts General Hospital (Boston, MA). EA is
defined as a tumor center located at or above the gastroesophageal
junction and had at least two-thirds of the bulk tumor located in the
esophagus; and BE is defined as pathologically confirmed intestinal
metaplasia . Controls were among healthy friends and non–
blood-related family members of hospitalized patients. Controls were
recruited at the same institutions in the same period as EA cases
. Pathologist-identified tissue regions that have more than 70%
tumor cells without definite evidence of necrosis are considered as
tumor tissues and used for DNA extractions. Peripheral venous blood
sample was drawn for eachsubject, andtheserum sample wasseparated
within 2 hours. The serum was isolated by centrifugation at 2000 rpm
for 10 minutes at 40°C and stored at −80°C until analysis. CfDNA
was extracted from 800-μl aliquots of serum using the Maxwell 16
blood DNA kit (Promega, Madison, WI). Tissue DNA was isolated
from 100 mg of EA tumor tissue by Maxwell 16 tissue DNA purifica-
Massachusetts General Hospital and Harvard School of Public Health
Genome-wide DNA Methylation Analysis
We used the Illumina Infinium HumanMethylation27 BeadChip
(Illumina, San Diego, CA) to analyze DNAm profiles. The BeadChip
contains 27,578 highly informative CpG loci covering more than
14,495 genes . DNA samples were bisulfite converted, then
whole-genome amplified (WGA), enzymatically fragmented, and
hybridized to the array. The assay was performed according to the
manufacturer’s instructions and was done at the BioMedical Genomics
Center, University of Minnesota.
After scanning of the BeadChip, data files were managed using the
Illumina BeadStudio software Methylation module. Each CpG site
on the BeadChip is represented by two bead types representing the
methylated (M) and unmethylated (U) state at that site. The methyla-
tion value for each CpG locus is expressed as a β value, representing
a continuous measurement from 0 (unmethylated) to 1 (completely
methylated) according to the following calculation: β value = (signal
intensity of M probe) / [(signal intensity of M + U probes) + 100];
the average β values is based on the average intensity of all U and
M CpG probes for a given locus.
Average β values were analyzed without normalization as recom-
mended by Illumina. The differences of DNAm levels (β value) be-
tween groups were analyzed using Student’s t test. Correlations of
mean DNAm levels of 27,578 CpG loci between tissue and serum
DNA samples were analyzed using the Pearson test. We selected the
most differentially methylated loci (a β value difference of >0.23 
and a P ≤ .0000006 between groups) to run hierarchical clustering
analysis. The choice of 0.23 as a criterion for a difference in β was
based largely on replicate experiments by Illumina that showed that
the HumanMethylation27 BeadChip could reliably detect a differ-
ence in β < 0.20 with a less than 1% false-positive rate . Un-
supervised hierarchical clustering analysis was carried out using the
R software program.
AverageDNAyields (range) were446.6ng(350.0-627.7ng),426.5 ng
(268.1-784.8 ng), 154.8 ng (145.1-172.2 ng), and 156.4 ng (128.2-
182.1 ng) for EA sera, matched EA tissue, BE sera, and control sera
samples, respectively. The OD 260/280 ratio, a measure of DNA
purity with respect to protein contamination, ranged from 1.32 to
1.80. Comparison of the number of CpG sites that could be success-
fully interrogated (as determined by BeadStudio software) revealed that
the call rates in both tissue DNA samples and sera DNA samples were
Figure 1. Correlation of DNAm levels (β values) between serum
DNA samples and matched tissue DNA samples (r = 0.92).
Genome-wide DNAm Profiling of cfDNAZhai et al.Neoplasia Vol. 14, No. 1, 2012
allgreater than 99.9%.Comparison ofβvaluedistribution between EA
sera DNA and matched tissue DNA showed similar patterns with high
peaks of hypomethylated loci and low peaks of the hypermethylated
loci (Figure W1). To investigate whether the methylation levels of sera
DNA were consistent with tissue DNA samples in EA, we carried out a
direct comparison of DNAm levels (β values) of 27,578 loci between
tissue DNA and matched serum DNA. Figure 1 shows that highly
correlated (r = 0.92) results can be achieved for matched tissue and
serum DNA, suggesting that genome-wide DNAm profiles in cfDNA
reflect DNAm profiles in tumor tissue DNA.
Clustering analyses showed that 911 loci perfectly discriminated
between EA and control samples, 544loci separated EAfrom BE sam-
ples, and 46 loci distinguished BE from control samples (Figure 2).
The large number of differentiated DNAm markers is consistent with
gene expression studies [33,34].
Genome-wide DNAm profiling has been performed successfully on
DNA recovered from several different types of tumor tissues includ-
ing ovarian , breast [31,36], prostate , and parathyroid 
cancers on the HumanMethylation27 BeadChip. Despite the robust
methylation profiling results from tumor tissues, little information
exists regarding the methylation analysis of cfDNA samples using
high-density methylation arrays. To our knowledge, this study is the
first to evaluate cfDNAm profiling on a genome-wide scale using the
Humanmethylation27 platform. Our data, although exploratory, sug-
gest that cfDNA obtained from serum can produce excellent DNAm
profiling on a genome-wide scale and may serve as a useful tool to
develop DNAm-based biomarkers for clinical application.
This study revealed several interesting observations. First, we ob-
served a high degree of concordance in DNAm profiles between
cfDNA and EA tumor tissue DNA on a genome-wide scale. This
proof-of-principle study suggests that a comprehensive analysis of
cfDNAm profiles has the potential to reflect genome-wide DNAm
alterations in primary tumor tissues. Second, we found that differen-
tial cfDNAm profiles can distinguish EA and BE from controls, as
well as EA from BE. The present results suggest that cfDNAm pro-
files may be a valuable biomarker for early detection of EA. Third, we
also observed a trend of increasing numbers of aberrant cfDNAm
markers with controls, BE, and EA (Figure 2). Our results are in
agreement with previous reports in which it was found that aberrant
tissues’ DNAm levels accumulated gradually with the histologic
changes from normal to BE, then from BE to esophageal carcinoma
[39–42]. These data suggest that cfDNAm profiles may reflect the in-
creasing involvement of aberrant DNAm in the process of normal-BE-
carcinoma sequence. However, because this is not a longitudinal study,
we cannot distinguish whether the abnormal cfDNAm markers seen
in the preneoplastic sera develop concurrently with adenocarcinoma,
or whether theaberrant cfDNAmmarkers inBE represent a predispos-
ing event that give rise to adenocarcinoma. Nevertheless, cfDNAm
profiles that might identify patients with cancer or at elevated risk
for developing cancer would have the potential to provide an opportu-
nity for early intervention. Furthermore, a benefit of using cfDNAm
marker is that serum can be easily obtained when biopsies are not avail-
able or when the exact position of the primary lesion is not clear. A
prospective longitudinal study should help reveal whether these aber-
rant cfDNAm profiles in normal or BE subjects are predictive of EA.
Our results should be viewed with caution. First, for the Human-
Methylation27 BeadChip, Illumina recommends a starting DNA
material of 500 ng or higher to achieve better performance. In this
study, the average DNA quantity was less than 500 ng and the overall
methylation marker completion rate was less than 100%, suggesting
that the starting DNA quantity of more than 500 ng is thus necessary
to obtain more robust methylation profiling results in future studies.
Moreover, the exploratory nature of this study is such that we are
unable to conduct sensitivity analysis and identify DNAm markers
number of tissue and serum samples from patients with EA. More works
in larger sample sizes are required to assess the roles of genome-wide
cfDNAm profiles in cancer diagnosis. Furthermore, cfDNAm profiling
MethyLight and MethDet platforms [43,44].
In conclusion, our data suggest that whole-genome amplified DNA
derived from stored serum have high potential to be used for genome-
wide methylation profiling. Genome-wide cfDNAm profiles may be a
useful noninvasive biomarker of EA and EA precursor lesions. Further
Figure 2. Hierarchical clustering of methylation values (β) from samples indicates that samples can be separated into distinct groups by
cfDNAm profiles. Columns represent samples. Rows represent CpG loci. Color represents methylation level (β) from 0 to 1 as per color
bar (red indicates low methylation level; yellow, high methylation level).
Neoplasia Vol. 14, No. 1, 2012Genome-wide DNAm Profiling of cfDNAZhai et al.
studies on cfDNAm are needed to compare the reproducibility of the
DNAm results obtained with different DNAm platforms and larger
numbers of samples. In addition, comparison of DNAm performance
between cfDNA obtained from fresh serum and DNA obtained from
archived serum samples stored in different environmental conditions
The authors thank the patients for their cooperation and participa-
tion in this study. We also thank Andrea Shafer and Salvatore Mucci
for data collection, entry, and management.
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_-FLA]_fig1",5,"place_anchor">_-FLA]_fig1",5,"pla- Download full-text
Figure W1. The β methylation value distributions in matched EA serum DNA (A) and tissue DNA (B).