DNA Methylation Changes in Atypical Adenomatous
Hyperplasia, Adenocarcinoma In Situ, and Lung
Adenocarcinoma
Suhaida A. Selamat1, Janice S. Galler1, Amit D. Joshi2, M. Nicky Fyfe3, Mihaela Campan1, Kimberly D.
Siegmund2, Keith M. Kerr3, Ite A. Laird-Offringa1*
1 Departments of Surgery and of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California,
Los Angeles, California, United States of America, 2 Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California,
United States of America, 3 Department of Pathology, Aberdeen Royal Infirmary, University of Aberdeen, Aberdeen, United Kingdom
Abstract
Background: Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor
development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural
history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation.
Methodology/Principal Findings: We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze
DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged
as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA
hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum
of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous
hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung
adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of
distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A
ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1,
NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML,
RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer.
Conclusions/Significance: The gradual increase in DNA methylation seen for numerous loci in progressively more
transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung
adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to
lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA
methylation changes might be used as molecular markers for the detection of preinvasive lesions.
Citation: Selamat SA, Galler JS, Joshi AD, Fyfe MN, Campan M, et al. (2011) DNA Methylation Changes in Atypical Adenomatous Hyperplasia, Adenocarcinoma In
Situ, and Lung Adenocarcinoma. PLoS ONE 6(6): e21443. doi:10.1371/journal.pone.0021443
Editor: Wael El-Rifai, Vanderbilt University Medical Center, United States of America
Received February 15, 2011; Accepted May 28, 2011; Published June 23, 2011
Copyright: ß 2011 Selamat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was funded by grants to Ite Laird-Offringa from Joan’s Legacy (now called Uniting Against Lung Cancer) and the Thomas G. Labrecque
Foundation, and NIH/NCI grant 5R01CA120869. The project was also supported in part by award number P30CA014089 from the National Cancer Institute. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely the responsibility of
the authors and does not necessarily represent the official views of the funding agencies.
Competing Interests: Ite A. Laird-Offringa is married to Peter W. Laird who is a consultant for Epigenomics, A.G., a company that is developing DNA methylation
assays for clinical use. The work described in this manuscript was performed without any support or involvement from Epigenomics. All authors declare that they
have no conflict of interest.
* E-mail: ilaird@usc.edu
than the conventional chest X-ray [5], and reports from studies
like the National Lung Screening Trial and the NELSON trial on
the effects of LDSCT screening on lung cancer mortality are
eagerly awaited [6,7]. However, even if mortality is reduced, this
imaging modality shows limited specificity; while LDSCT can
detect stage I cancers, a number of these may not actually progress
to late stage cancer [8]. Thus, in order to avoid unnecessary
interventions, we must gain better insight into the molecular
changes underlying the natural history of lung cancer. Such
knowledge could be used to develop additional molecular tests that
might complement LDSCT screening, allowing detection of those
Introduction
Lung cancer is the leading cause of cancer-related death in the
world, and is estimated to have caused over 1.3 million deaths in
2008 [1,2]. World wide, smoking accounts for 80% of all lung
cancer deaths in males and 50% of those in females [3]. The
overall five-year survival of patients with lung cancer is very poor;
in the United States it is a dismal 18% despite extensive efforts to
improve diagnosis and treatment [4]. Sensitive new visual
diagnostic modalities such as low dose spiral computed tomography (LDSCT) show potential to detect much smaller lung lesions
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
lesions that would progress to tumors with metastatic potential.
The analysis of DNA methylation might provide such a test.
Abnormal DNA methylation is an epigenetic change that has been
widely observed in all types of cancer including lung cancer [9–
11]. It consists of the addition of a methyl group to the 5-position
of cytosine in the context of a two-base pair palindrome, or CpG
dinucleotide. Sensitive molecular assays allow detection of DNA
methylation in tumors as well as in patient bodily fluids [11–14],
and it therefore holds much promise as a possible molecular
marker to complement image-based lung cancer screening.
This study focuses on lung adenocarcinoma, a histological
subtype of lung cancer that is increasing in many countries [15–
19], and which currently accounts for at least 37% of all lung
cancer in the United States [4]. While smoking remains the
predominant cause of lung adenocarcinoma, this histological
subtype is also the most common form of lung cancer amongst
never smokers, Asians and women [4,20]. Unlike squamous cell
carcinoma, the natural history of lung adenocarcinoma is still
poorly understood. Studies suggest that at least some lung
adenocarcinomas arise from preneoplastic lesions called atypical
adenomatous hyperplasia (AAH), which progress to adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar
carcinoma or BAC), and eventually develop into invasive cancer
[21–24]. In 1999, the WHO acknowledged AAH as a putative
preneoplastic lesion of lung adenocarcinoma. AAH is now defined
as ‘‘localized proliferation of mild to moderately atypical cells
lining involved alveoli and sometimes respiratory bronchioles,
resulting in focal lesions in peripheral alveolated lung, usually less
than 5 mm in diameter’’ [25]. AIS is defined as ‘‘a localized small
(,3 cm) adenocarcinoma with growth restricted to neoplastic cells
along preexisting alveolar structures (lepidic growth), lacking
stromal, vascular, or pleural invasion. Papillary or micropapillary
patterns and intraalveolar tumor cells are absent.’’ [24]. AIS is
very rarely mucinous. It is associated with a 100% five year postresection patient survival, and is similar in morphology to highgrade AAH lesions. Both AAH and AIS can be found as incidental
findings in the lungs of patients resected for a primary lung tumor,
usually adenocarcinoma [21]. However, with the advent of more
sensitive radiological imaging, these lesions are now being
individually detected using fine section high resolution computed
tomography [26,27]. A number of molecular studies support the
existence of an AAH-AIS-adenocarcinoma continuum [9]. LOH
events at 9q and 16p, key features of lung cancer, have been
reported to occur at similar frequencies in AAH and adenocarcinoma [28,29], and the mutually exclusive natures of KRAS and
EGFR mutations reported in lung adenocarcinoma are maintained
in AAH lesions [30]. Support for a developmental sequence from
AAH to adenocarcinoma also comes from conditional oncogenic
mouse models for lung adenocarcinoma, in which KRAS or EGFR
genes are activated. In both types of mice, AAH-like lesions are
found before the emergence of adenocarcinomas [31,32].
Abnormal DNA methylation has not yet been thoroughly
examined in AAH and AIS. Extensive investigation of DNA
methylation in AAH has been impeded by the minute size of these
lesions and the necessity to use bisulfite conversion. This chemical
treatment specifically deaminates unmethylated cytosine to uracil,
but not 5-methylated cytosine, thereby embedding DNA methylation information into the DNA sequence. Unfortunately, bisulfite
treatment can result in considerable degradation of already scarce
genetic material [33]. To date, DNA methylation analysis of AAH
has required the use of multiplexed nested methylation-specific
polymerase chain reaction (MS-PCR), disallowing quantitative
assessment of DNA methylation and limiting the number of genes
that can be tested [34,35]. In this study, we overcame these
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limitations by using the sensitive technology MethyLight, which
consists of real-time PCR of bisulfite-converted DNA, using
primers and probes designed to specifically hybridize to methylated regions that retained cytosines [14]. We used MethyLight to
successfully quantitatively assess DNA methylation levels at 15
CpG islands prone to hypermethylation in lung adenocarcinoma,
and also assessed global hypomethylation by examining DNA
methylation of repeated sequences. We examined DNA methylation in tissue samples spanning the putative spectrum of
peripheral lung adenocarcinoma development: histologically
normal adjacent non-tumor lung from non-lung cancer patients
as well as lung cancer patients, AAH, AIS, and invasive lung
adenocarcinoma (Figure 1).
Because AAH lesions provide very little DNA, we carefully
weighed our choice of the loci to interrogate, selecting 15 loci that
constitute strong candidates for non-invasive DNA methylation
markers for lung adenocarcinoma detection. From a prescreening
of 114 loci, we had previously chosen 28 that were most
differentially methylated between tumor and adjacent non-tumor
lung for further analysis and had identified 12 of these loci as
significantly hypermethylated in lung adenocarcinoma [36]. We
have subsequently evaluated hundreds more loci (using individual
probes, a CpG island microarray, and an Illumina GoldenGate
analysis), yielding additional loci highly and frequently methylated
in lung adenocarcinoma (unpublished information). From our
cumulative data sets, we chose the 15 loci that showed the most
promise for development into molecular markers for lung
adenocarcinoma (Table S1). These loci are also of interest for
the potential biological implications of their DNA methylation.
Fourteen of these represented loci that were very frequently and
highly methylated: 2C35, CDH13, CDKN2A ex2, CDX2, EYA4,
HOXA1, HOXA11, NEUROD1, NEUROD2, OPCML, PTPRN2,
SFRP1, TMEFF2 and TWIST1. We added RASSF1 because we
had observed that, although its methylation frequency is not as
high in adenocarcinoma as some other loci [36,37], it can be
methylated in those adenocarcinomas showing little DNA
methylation of the other commonly methylated loci, in other
words, its DNA methylation profile can be complementary [36].
We have validated these 15 CpG islands as being significantly
hypermethylated in lung adenocarcinoma compared to adjacent
non-tumor lung in two additional independent sample collections
([36] and unpublished results).
Besides local hypermethylation at CpG islands, global DNA
hypomethylation is also a hallmark of cancer, and is associated with
retrotransposon activation and genomic instability [38]. In order
to examine hypomethylation in our analysis, we included two
repeat-based DNA methylation probes (SAT2-M1 and ALU-M2
[39]) in the study. The mean methylation of these two probes has
been shown to correlate well with global DNA methylation levels
[39]. Thus, our selection of probes was tailored to provide key
insights into the occurrence of DNA methylation alterations in
putative precursor lesions to lung adenocarcinoma.
Methods
Ethics statement
All human tissue samples were paraffin-embedded archival
remnants of tissue resected for clinical purposes, and were
obtained from Aberdeen University Medical School. The research
was approved as exempt from the need to obtain informed consent
by the USC IRB (# HS-CG-07-00017) and by the Grampian
Research Ethics Committee (study 05/S0801/141). The latter
body stipulates that no consents are required from deceased
subjects or when de-identified remnants of archival tissue are used.
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
Figure 1. Lung and lesion histology. Haematoxylin and eosin-stained sections of (A) Adjacent non-tumor lung (B) Atypical adenomatous
hyperplasia (C) Adenocarcinoma in situ (D) Lung adenocarcinoma, all at 1006 magnification.
doi:10.1371/journal.pone.0021443.g001
Pathology, Aberdeen Royal Infirmary, prospectively and specifically examines all surgical lung resection specimens received for
AAH and AIS lesions, as well as the index lesion that requires
surgical resection. All specimens are inflated per-bronchially with
10% neutral buffered formalin and cut into 1 cm thick parasagittal
sections after a 24-hr fixation. All visible lesions of .1 mm
diameter are sampled. In addition up to 6 random parenchymal
tissue blocks are taken from the lung surrounding but separate
from the main lesion, which is most often a primary carcinoma. A
minority of lesions is visible to the trained naked eye on such gross
examination of the lung slices; most AAH lesions are only detected
at microscopy. This approach has provided a high yield of both
AAH and AIS lesions over many years [21]. As additional controls
we also analyzed 30 independent samples of histologically verified
The identities of the subjects were unknown to USC investigators
or lab personnel.
Study subjects
Information on the subjects from whom the samples were
procured is provided in Table S2. Because of the archival nature
of the samples, and the fact that many patients had long since been
deceased, very limited smoking information was available. The
distribution of AdjNTL, AAH, AIS and adenocarcinoma tissue
samples derived from 63 subjects is described in Tables 1 and 2.
AAH lesions are generally quite difficult to find. In order to
identify such lesions with more frequency the Department of
Table 1. Distribution of AdjNTL, AAH, AIS and
adenocarcinoma samples among 63 subjects.
Lesion Type:
AdjNTL
AAH
N = 101
+
+
N = 31
+
+
N = 19
+
N = 18
+
N=3
+
N = 10
+
Number of subjects
with samples of
each type2
63
AIS
Table 2. Distribution of multiple lesions among the cases.
Adenocarcinoma
Number of each type
of lesion obtained from
a single subject
+
Subjects
with AAH
Subjects
with AIS
Subjects
with AD
+
1
23
11
48
+
2
8
2
2
+
+
3
7
0
0
+
+
+
4
2
1
0
41
16
50
5
1
1
0
6
0
0
0
7
0
1
0
Total subjects
41
16
50
Total number of lesions1
73
31
52
+
1
13 subjects lacked an adenocarcinoma sample either because it was no longer
available (4) or because the patient had a different type of lung cancer (1
mixed adeno/squamous, 5 large cell carcinomas, 2 squamous cell cancers and
1 carcinoid).
2
Total number of subjects from which AdjNTL, AAH, AIS or adenocarcinoma was
studied: 63. MetNTL was obtained from an additional 30 subjects.
doi:10.1371/journal.pone.0021443.t001
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1
In addition, a single AdjNTL was obtained from each of 63 subjects and a single
MetNTL sample was obtained from each of 30 subjects.
doi:10.1371/journal.pone.0021443.t002
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
cancer-free lung (MetNTL, see Table S2) from non-lung cancer
patients who had been operated for a single pulmonary metastasis
from a different organ site (usually colorectal cancer). In total, 249
formalin-fixed paraffin-embedded tissues from 93 subjects were
included in the statistical analyses.
approach that allows us to use all lesions of the same type from the
same individual in the analysis, while properly accounting for the
possible within-individual correlation in DNA methylation values.
We first verified that the data satisfy the assumption that the
average DNA methylation value was the same in lesions from
patients with one lesion compared to patients with multiple lesions
of the same type (data not shown). For each marker, two groups
were then compared by regressing the rank of the PMR values on
an indicator variable for group membership. The rank transformation was used to address skewness in the PMR value when
testing for differences in group means, ranking all 249 samples
before proceeding with the pair-wise group comparisons. Hypothesis testing used robust variance estimates under an independence
working correlation structure. All testing was performed at the 5%
significance level.
To identify in which lesion type, AAH, AIS, or adenocarcinoma, the markers first showed a difference in average DNA
methylation value, we performed a series of univariate tests,
comparing DNA methylation values between pairs of histologies:
AdjNTL vs. AAH, AAH vs. AIS, and AIS vs. adenocarcinoma. To
account for conducting three tests for each marker (multiple
testing), we applied a Bonferroni correction to determine statistical
significance, requiring a cutoff of p,0.017 ( = 0.05/3 tests) for
statistical significance. Markers were classified into the categories
‘‘early’’, ‘‘intermediate’’, or ‘‘late’’, depending on the pairwise
comparison that yielded the first increase in average DNA
methylation value that both achieved statistical significance, and
showed a group median of .1 on the raw PMR scale. The PMR
scale runs from 0 to 100 (100 indicates complete methylation
compared to enzymatically methylated human DNA); a .1 PMR
cut-off was chosen to minimize undue emphasis on very low levels
of DNA methylation that are not likely to be biologically
significant. Following this analysis, we investigated the potential
for a ‘‘field defect’’ in the lung by comparing DNA methylation
values in AdjNTL with MetNTL. As none of the 15 hypermethylation markers or the hypomethylation measure had been
compared previously between these two tissue types, we controlled
for multiple testing by requiring a Bonferroni-corrected p-value
(p,0.0031 = 0.05/16 tests) to declare statistical significance.
We performed a cluster analysis to see if we could identify any
subgroups within AAH lesions. Using the 15 hypermethylation
loci, we applied partitioning around medoids (PAM) [46], using
silhouette width to select the number of clusters. For all markers,
DNA methylation values were compared between the two
identified clusters using GEE and a Bonferroni cutoff of
p,0.0033 (15 tests). The same methods were applied to compare
AAH lesions based on histologic grade: high grade (HG) and low
grade (LG).
To examine the potential effects of clinical variables on the
analysis, we used ANOVA (for age and packyears) and the Chisquare test (for gender and known smoking status) to examine
whether these variables differed significantly between sample types
(MetNTL, AdjNTL, AAH, AIS, adenocarcinoma). Statistical
analyses were performed using STATA version 10, Prism 5, and
R.2.10.0.
DNA extraction and bisulfite treatment
Each section was hematoxylin stained and evaluated by an
experienced pathologist (KMK), who carefully marked the lesions
to be retrieved. Slides were manually microdissected under the
microscope and DNA was extracted by proteinase K digestion.
Microdissected cells were incubated overnight at 50uC in a buffer
containing 100 mM TrisHCl (pH 8.0), 10 mM EDTA (pH 8.0),
1 mg/ml proteinase K, and 0.05 mg/mL tRNA. Extracted DNA
was bisulfite converted using Zymo EZ DNA Methylation kit
(Zymo Research, Orange, CA) with a modification to the protocol
in which samples were cycled at 90uC for 30 seconds and then
50uC for one hour, for up to 16 hours total. Bisulfite-treated DNA
was subjected to quality control tests for DNA amount and
bisulfite conversion [40]. DNA levels were determined by a
bisulfite conversion-independent ALU reaction (ALU-C4), consisting of a primer/probe set lacking CpGs [40]. A conservative cutoff
was set at Ct (threshold cycle) #22 after extensive analyses
comparing data with a cutoff of ALU Ct #20 with that of Ct #22
showed no statistically significant difference in percentage
methylated reference (PMR) values (see below) between the two
(data not shown). In addition, a previous study demonstrated that
samples with Ct values #24 still yielded reliable results [41]. Four
independent AAH samples with ALU Ct values .22 were thus
excluded.
DNA methylation analysis
Bisulfite-treated DNA was analyzed by MethyLight as described
[42]. Primer and probe sequences are listed in Table S1. Locus
2C35 was identified by restriction landmark genomic sequencing
to be highly methylated in non-small cell lung cancer [43] as well
as other types of cancer [44]. The CDKN2A ex2 primer/probe set
detects highly significant hypermethylation in a CpG island in
exon 2 of CDKN2A in lung adenocarcinoma vs. adjacent nontumor lung, showing more highly significant differences than
probes for upstream CpG islands [36]. The OPCML primer/probe
set also targets the CpG island of the adjacent and closely related
family member HNT [36]. The SAT2 and ALU probes (SAT2-M1
and ALU-M2) DNA methylation values were averaged and used as
an indicator for global DNA methylation levels [39]. ALU-M2 is
distinct from the ALU-C4 probe that hybridizes to a methylationindependent (CpG-less) region of ALU repeats and that was used
for input DNA normalization [40]. Genomic DNA which was
exhaustively enzymatically methylated by three consecutive M.SssI
treatments was used as a reference sample to generate standard
curves. MethyLight data is represented as the percentage
methylated reference (PMR), which is defined by the GENE:
ALU-C4 ratio of a sample, divided by the GENE: ALU-C4 ratio of
M.SssI-treated reference DNA [40]. While it is rare, occasionally
PMR values of more than 100 can be observed, indicating that the
reference DNA might not be fully methylated at a particular site.
The same batch of reference DNA was used throughout this study
to avoid any bias.
Results
Statistical analyses
DNA methylation levels across the AdjNTL-AAH-AISadenocarcinoma spectrum
We included a total of 249 tissue samples from 93 subjects in the
analysis. Our primary statistical analysis was to compare the DNA
methylation values between groups of different lesion types using
generalized estimating equations (GEE [45]). GEE is a regression
We used a comprehensive collection of tissues encompassing
adjacent non-tumor lung (AdjNTL), the putative adenocarcinoma
precursor lesions AAH and AIS, as well as synchronous
adenocarcinoma (Tables 1 and 2). Since AdjNTL from lung
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
were designated as ‘‘early’’ loci, with statistically significantly
higher DNA methylation in AAH than in AdjNTL (Figure 3).
PTPRN2 showed a further significant increase in DNA methylation in adenocarcinoma vs. AIS (the increase from AAH to AIS
did not meet our multiple comparisons threshold). Seven loci,
2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and
TMEFF2, were designated as ‘‘intermediate’’, or characteristic
for AIS (Figure 4). Significant DNA hypermethylation of these loci
was observed in AIS compared to AAH, and for four of these loci,
DNA methylation levels further increased significantly in adenocarcinoma compared to AIS. Five remaining loci, CDH13, CDX2,
OPCML, SFRP1 and TWIST1 were designated as ‘‘late’’ loci;
significantly elevated DNA hypermethylation was only detected in
adenocarcinoma, as compared with AIS (Figure 5). RASSF1
hypermethylation approached significance but did not meet our
multiple comparisons cut-off in the AIS to adenocarcinoma
comparison. However, RASSF1 was highly significantly hypermethylated in adenocarcinoma vs. AdjNTL, and the scatterplot
supports the notion that RASSF1 hypermethylation is a late event
(Figure 5). Significant DNA hypermethylation of these six loci
would therefore appear to be associated with invasive lung
adenocarcinoma. Examination of the mean of the two repeat
probes as an indicator of global DNA hypomethylation showed
highly significant hypomethylation only in the AIS to adenocarcinoma comparison (Figure 6) suggesting that global DNA
hypomethylation may be a late event in lung adenocarcinoma
development.
Baseline DNA methylation levels in AdjNTL were in general
low, however, modest methylation was observed for several of the
15 DNA hypermethylation markers (Table 3). To determine
cancer patients might show DNA methylation ‘‘field defects’’ and
general molecular changes arising from environmental exposures
such as tobacco smoke [47], we included adjacent lung tissue from
resections of 30 subjects with single pulmonary metastases from
non-lung primary cancers (MetNTL) in the study. Our sample
collection also included cases in which multiple AAH and AIS
lesions were obtained from a single subject (Table 2), which
allowed evaluation of the spectrum of DNA methylation changes
within individuals. Each of the AAH and AIS specimens was
pathologically confirmed to be an isolated lesion separate from any
other lesions in the same lung.
We had previously found all 15 CpG islands to be highly
significantly methylated in lung adenocarcinoma compared to
AdjNTL (tissues derived from lung cancer patients from the Los
Angeles area, the East coast of the United States, and Ontario,
Canada ([36] and unpublished data). Here, we confirmed these
findings, observing highly significant DNA hypermethylation in
adenocarcinoma vs. AdjNTL for all 15 hypermethylation loci (all
p,161025, Table 3) in samples originating from the United
Kingdom. This indicates that lung adenocarcinoma samples from
a variety of geographic areas can exhibit similar hypermethylation
profiles.
We next determined whether these DNA methylation changes
are present in the presumptive precursor stages of the disease,
AAH and AIS (Figure 2 and Table 3). Markers were classified into
the categories ‘‘early’’, ‘‘intermediate’’, or ‘‘late’’, depending on
the pairwise comparison that yielded the first increase in average
DNA methylation value that both achieved statistical significance,
and showed a group median of .1 on the raw PMR scale
(Table 3). According to these criteria, CDKN2A ex2 and PTPRN2
Table 3. Median Percentage Methylated Reference (PMR) and pair-wise comparison p-values between each tissue type.
p-values for pair-wise comparisons of tissue types1
Median PMRs
MetNT
(n = 30)
AdjNT
(n = 63)
AHH
(n = 73)
AIS
(n = 31)
AD2
(n = 52)
CDKN2A EX2
4.5
5.0
11
19
21
PTPRN2
3.7
1.1
2.8
8.8
2C35
0.53
0.60
1.1
EYA4
2.0
1.6
Locus
MetNT vs.
AdjNTL
AdjNTL
vs. AAH
AAH
vs. AIS
AIS vs.
AD
AdjNTL
vs. AD
0.0031
0.017
0.017
0.017
0.05
0.42
2.6E-11
0.045
0.90
,2.1E-14
Early
19
7.7E-5
2.1E-3
0.023
5.5E-3
,2.1E-14
Early
11
24
0.54
0.18
7.2E-4
0.032
,2.1E-14
Interm
0.41
3.1
16
0.94
0.092
7.0E-4
1.5E-4
1.3E-11
Interm
BH p-value threshold
Designation
HOXA1
,0.01
0.015
0.12
4.6
21
0.15
0.16
8.7E-5
0.037
,2.1E-14
Interm
HOXA11
1.5
0.92
1.3
7.8
19
0.014
0.23
6.7E-8
1.4E-4
,2.1E-14
Interm
NEUROD1
0.29
0.17
0.60
3.9
13
0.18
0.014
0.011
3.1E-3
,2.1E-14
Interm
NEUROD2
0.78
1.3
1.2
4.0
12
0.0056
0.71
0.016
7.1E-3
,2.1E-14
Interm
TMEFF2
6.1
4.9
6.1
19
18
0.089
0.19
1.9E-9
0.23
8.9E-7
Interm
CDH13
,0.01
,0.01
0
,0.01
1.3
0.89
0.039
4.8E-3
5.5E-10
2.1E-14
Late
CDX2
1.3
0.46
0.53
1.6
10
0.17
0.94
0.061
6.1E-3
,2.1E-14
Late
OPCML/
HNT3
0.32
0.028
0
0.29
5.3
0.097
0.067
1.5E-3
3.0E-4
,2.1E-14
Late
RASSF1
0.44
0.12
,0.01
0.23
8.9
8.0E-4
0.0090
9.0E-3
0.026
8.5E-6
Late
SFRP1
0.29
0.44
0.15
0.29
8.0
0.27
0.071
0.52
6.9E-12
2.9E-14
Late
TWIST1
0.010
,0.01
0
0.12
16
0.40
4.4E-3
4.2E-3
9.9E-3
,2.1E-14
Late
Mean Repeats
80
71
72
75
46
0.076
0.12
0.99
1.0E-9
2.4E-9
Late
1
p-values are from GEE analysis. Designations of ‘‘early’’ ‘‘intermediate’’ and ‘‘late’’ are based on statistically significant p-values with the restriction that any locus
designated as hypermethylated have a median PMR value of $1 (bolded). This was done to minimize attributing significance to biologically meaningless differences.
AD = adenocarcinoma.
3
The OPCML/HNT primer/probe set recognizes two adjacent CpG islands for the homologous OPCML and HNT genes.
doi:10.1371/journal.pone.0021443.t003
2
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
Figure 2. Heatmap of DNA methylation levels of 15 loci and repeats in all tissue types. Loci are arranged in alphabetical order. Dark blue
indicates very low levels of DNA methylation, yellow indicates high levels of DNA methylation, and missing values are indicated in white. The type of
sample is indicated at the top.
doi:10.1371/journal.pone.0021443.g002
indicative of their propensity to progress. In order to assess the
existence of any sub-groups of preneoplastic lesions differing in
DNA methylation profiles, we examined the relationship of the
samples and the 15 DNA hypermethylation probes using
partitioning around medoids (PAM). We observed two distinct
clusters of 68 and 5 samples. In the latter group, the five AAH
lesions from four individuals had statistically significantly higher
DNA methylation levels for 2C35, CDKN2A ex2, CDX2, HOXA1,
NEUROD1, TMEFF2 and TWIST1 than the remaining 68 samples
(all p,0.003).
AAH lesions are sometimes divided into high grade (HG) and
low grade (LG) based on histology. However, this distinction can
be rather subjective. The grade determination did not correlate
with our delineation of the two AAH clusters. We compared PMR
values from AAH lesions histologically denoted as high-grade
(HG, n = 11) to low-grade (LG, n = 45) lesions and found no
statistically significant differential DNA methylation between the
two histologies after multiple comparison correction (Table S3).
whether these potentially elevated DNA methylation levels could
be an indication of a ‘‘field defect’’, we compared DNA
methylation levels of the 15 hypermethylation probes and the
global DNA methylation measure in AdjNTL vs. MetNTL. Only
one hypermethylation locus met the criterion for a statistically
significant difference in methylation between the two tissue types:
PTPRN2 (Table 3). DNA methylation levels for PTPRN2 were
lower in AdjNTL compared to MetNTL (median PMR of 1 vs. 4),
not higher. This difference is difficult to discern from Figure 3 due
to low variation in PMR values (interquartile ranges of 0.4–3.3 for
AdjNTL and 2.0–5.5 for MetNTL) and the scale on the vertical
axis. PTPRN2 also showed significantly increased DNA methylation from AIS to adenocarcinoma. Thus, we did not find elevated
DNA methylation in AdjNTL compared to MetNTL for any
locus, nor did we observe any significant difference in global
hypomethylation (Table 3, bottom row).
With the limited smoking information we had, we examined
whether smoking status (current or past) or packyears of smoking
were associated with DNA methylation levels seen in AdjNTL, and
might explain the variability seen in baseline DNA methylation
levels. We observed no significant differences (data not shown).
Discussion
Our observation that distinct loci show DNA hypermethylation
at different stages of the putative adenocarcinoma development
sequence and that the number of methylated loci and DNA
methylation levels are generally higher in each progressive stage,
support a model in which AAH and AIS are precursor stages of at
Analysis of DNA methylation in preneoplastic lesions
It has been proposed that not all AAH lesions progress to
cancer. If true, some AAH could show molecular changes
Figure 3. ‘‘Early’’ DNA methylation changes: scatterplots of loci significantly hypermethylated in AAH lesions compared to AdjNTL.
p-values were calculated by GEE, with a Bonferroni cutoff of p,0.017 (see Methods). Statistically significant differences are marked with an asterisk.
Interquartile ranges are marked with red bars.
doi:10.1371/journal.pone.0021443.g003
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
Figure 4. ‘‘Intermediate’’ DNA methylation changes: scatterplots of loci significantly hypermethylated in AIS lesions compared to
AAH. p-values were calculated by GEE, with a Bonferroni cutoff of p,0.017 (see Methods). Statistically significant differences are marked with an
asterisk. Interquartile ranges are marked with red bars.
doi:10.1371/journal.pone.0021443.g004
Figure 5. ‘‘Late’’ DNA methylation changes: scatterplots of loci significantly hypermethylated in adenocarcinoma compared to AIS.
p-values were calculated by GEE, with a Bonferroni cutoff of p,0.017 (see Methods). Statistically significant differences are marked with an asterisk.
Interquartile ranges are marked with red bars. RASSF1 was included in the figure because hypermethylation is clearly present increased
adenocarcinoma, although the AIS vs. adenocarcinoma comparison did not reach statistical significance (p = 0.026, see Table 3).
doi:10.1371/journal.pone.0021443.g005
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
noma continuum. A longitudinal study, in which lesions are
studied over time in the same individual, would be the best way to
study the natural history of cancer, but this is very difficult to do
for peripheral lung cancer given the small size and inaccessibility
of preinvasive lesions. Because our study was cross-sectional,
comparing individual lesions from a collection of patients, any
temporal interpretations should be treated with caution; the results
could be affected by confounding factors such as age, gender and
smoking history. Examination of gender and age showed no
significant differences between AdjNTL, AAH, AIS and adenocarcinoma groups, nor did we find a relationship between smoking
status (current of former) or packyears and DNA methylation
levels. However, the number of subjects for which any smoking
information was available was small (n = 19). To further support
our findings, we therefore also examined two subsets of samples
from our collection: the samples from the 10 subjects from whom
AdjNTL, AAH, AIS, adenocarcinoma were all available (top row,
Table 1), and the collection of samples obtained from 16
confirmed current or previous smokers with at least 20 packyears
or more of smoking. While the two subsets had a much smaller
sample size and therefore had less power than the full collection,
we observed that the hypomethylation measure and the majority
of hypermethylation loci (10/15) showed similar changes in
median PMR and would classify to the same category (early,
intermediate or late) in both subsets, either through statistical
significance or trending to statistical significance (data not shown).
This suggests that our observations are robust and not the result of
confounding factors.
Of the 15 loci we studied, the CpG islands of CDKN2A ex2 and
PTPRN2 are the only two that we found to be significantly
hypermethylated in AAH lesions compared to adjacent non-tumor
Figure 6. Global DNA methylation levels in AAH, AIS, and lung
adenocarcinoma. The average of ALU-M2 and SAT2-M1 probes were
used as indicators of global DNA methylation. p-values were calculated
by GEE, with a Bonferroni cutoff of p,0.017 (see Methods). Statistically
significant differences are marked with an asterisk. Interquartile ranges
are marked with red bars.
doi:10.1371/journal.pone.0021443.g006
least a subset of lung adenocarcinomas. The data indicate that
distinct epigenetic events occur with the transition to hyperplasia,
carcinoma in situ and finally invasive cancer (summarized in
Figure 7) and imply a model similar to that for the development of
colorectal and breast cancers [48–51]. Our quantitative observations build on previous reports of increased DNA methylation
frequency in AAH compared to adjacent non-tumor tissue [34,35]
and suggest that this trend continues in the AIS to adenocarci-
Figure 7. Summary of DNA methylation changes in AAH, AIS, and lung adenocarcinoma. The putative sequence of DNA
hypermethylation events is indicated by the color shading and position of locus names. Dark shading indicates hypermethylation. Global DNA
hypomethylation is only significantly altered in the AIS to adenocarcinoma comparison, though it appears to occur sporadically even in histologically
normal tissue.
doi:10.1371/journal.pone.0021443.g007
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
lung. Frequent deletions and mutations of CDKN2A (a negative
regulator of cell cycle, also known as p16) in lung cancer were first
observed in 1994 [52] and hypermethylation and silencing was
subsequently observed to occur in substantial numbers of cancers
carrying an intact gene [53]. Inactivation of CDKN2A by DNA
hypermethylation is now thought to be one of the earliest events
during lung cancer development ([54] and references therein) and
is observed in hyperplasia and carcinoma in situ [34,55,56]. We
focused on the exon 2 CpG island of the gene because our
previous study of CDKN2A DNA methylation showed that it was
more highly significantly associated with cancer compared to
adjacent non-tumor lung than the promoter CpG island.
However, it should be noted that some cancer cell line data
suggests that CDKN2A exon 2 DNA methylation is not necessarily
associated with gene silencing [57]. Methyl-binding protein
MeCP2 has been shown to associate with methylated CDKN2A
exon 2, but the biological significance of this modification for
cancer progression remains to be clarified [57]. The functional
consequences of CDKN2A exon 2 DNA methylation in tumor
samples merits further investigation. Interestingly, CDKN2A
hypermethylation at the promoter CpG island been associated
with progression of stage 1 lung cancer [58], suggesting the
importance of continued inactivation of this gene during
progression.
Little is known about the function of PTPRN2, a receptor type
protein tyrosine phosphatase (PTP) that is a major autoantigen in
insulin-dependent diabetes mellitus [59] and that is also expressed
in the cerebellum and other parts of the nervous system [60].
Because PTPs dephosphorylate proteins, many of these enzymes
are implicated in the negative regulation of cell growth,
differentiation and oncogenic transformation [61]. A variety of
PTPs have been shown to be mutated in colorectal cancer [62]
and PTP receptor-type D was identified as mutated and
inactivated in lung adenocarcinoma [63]. We have found PTPRN2
to be frequently methylated in adenocarcinoma and squamous cell
cancer of the lung, in at least two independent sample sets for both
histological subtypes ([64] and unpublished results). However,
functional studies on the potential role of this protein in any type
of cancer remain to be done.
We observed significant DNA hypermethylation in AIS
compared to AAH for seven loci: 2C35, EYA4, HOXA1, HOXA11,
NEUROD1, NEUROD2 and TEMFF2. 2C35 was identified
through restriction landmark genomic scanning to be hypermethylated in lung cancer [43] as well as in primitive neuroectodermal
tumors, gliomas and colon cancer, and these observations were the
basis for the design of MethyLight probe/primer set and our
examination of this locus in lung cancer. The CpG island does not
overlap with a known gene, although it overlaps with an
uncharacterized expressed sequence tag (DA773580 [65]). The
nearest identified gene, located 20 kb downstream, is PTF1A,
pancreas specific transcription factor 1a, a helix-loop-helix
transcription factor promoting acinar differentiation in the
pancreas and showing loss of function in pancreatic cancer [66].
To date no role of PTF1A in lung cancer has been reported. Thus,
the biological relevance of DNA methylation at 2C35 remains to
be investigated. One possibility is that this locus carries an
enhancer that might normally drive the expression of one or more
distant genes; in human H1 embryonic stem cells the region
containing 2C35 shows histone 3 lysine 4 mono-methylation, a
mark that is associated with enhancers and regions downstream of
transcription start sites (data from the Bernstein lab at the Broad
Institute, [67]). EYA4, the human homologue for eyes-absent 4
from Drosophila, is a tyrosine phosphatase that targets histone
H2AX and plays a role in recruiting the DNA repair machinery to
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DNA. The gene is inactivated by DNA methylation in Barrett’s
esophagus and esophageal adenocarcinoma [68]. We found
significant DNA hypermethylation of both HOXA1 and HOXA11,
which lie about 90 kilobases apart at opposite ends of the HOXA
cluster, in AIS. HOX genes have been reported to be coordinately
hypermethylated in lung cancer, particularly adenocarcinoma
[69,70]. In breast cancer, HOXA1 was identified as a frequently
methylated, and in an analysis similar to ours, was found to be
significantly hypermethylated in atypical ductal hyperplasia (ADH)
relative to normal breast, and ductal carcinoma in situ (DCIS)
relative to ADH [50]. However, no multiple comparisons
correction was applied in the latter study; using such a correction
HOXA1 is only significantly hypermethylated in DCIS vs. ADH,
which is very similar to our finding of significant hypermethylation
in AIS compared to AAH. TMEFF2, a transmembrane protein
with EGF-like and two follistatin-like domains (also known as
hyperplastic polyposis protein (HPP1) and tomoregulin), was found
to be similarly hypermethylated in DCIS in the breast cancer
study. TMEFF2 had previously been reported to be methylated in
lung adenocarcinoma [71], and inactivation of DNA methyltransferase 1 in a breast cancer cell line reactivates methylated
TMEFF2 [72], suggesting its DNA methylation leads to silencing.
We thus identified two loci, HOXA1 and TMEFF2, that appear to
have an ‘‘intermediate’’ role in cancer development in the lung as
well as the breast. NEUROD1 and 2 were identified by us as highly
methylated in lung adenocarcinoma compared to AdjNTL.
NEUROD1 DNA methylation has been observed in diffuse large
B-cell lymphoma [73] and breast cancer where it was associated
with a ten-fold more likely response to neoadjuvant therapy in
estrogen receptor-negative cancers [74]. It is intriguing that just
like PTPRN2, NEUROD proteins appear to be involved both in
diabetes mellitus and cerebellar development [75].
CDH13, CDX2, OPCML, SFRP1 and TWIST1 do not show
significant hypermethylation in AAH or AIS, and instead are only
significantly hypermethylated in invasive adenocarcinoma. Inactivation or hypermethylation of many of the latter genes has been
linked to poor prognosis or metastasis, agreeing with a potential
role in the development of invasive cancer. CDH13 or heart
cadherin, encoding and adhesion molecule, was identified as DNA
hypermethylated in lung cancer in 1998 [76], a finding that was
substantiated by many studies (e.g. [36,77–80]). CDH13 DNA
methylation has been found to be associated with stage IV disease
[81], poor prognosis [82], and tumorigenicity of xenografts in
nude mice [83]. In a silica-induced lung cancer animal model,
CDH13 DNA methylation was seen in invasive but not preinvasive
lung cancer [84], and in an analysis of stage I lung cancer patients,
it was observed to be associated with recurrent cancer [58]. Thus,
loss of CDH13 may be linked to the altered adhesive properties
that allow cells to become invasive. Likewise, OPCML, an opioid
receptor and putative tumor suppressor thought to play a role in
adhesion [85], appears to become DNA methylated late, showing
hypermethylation mainly in adenocarcinoma. Silencing of OPCML
has been implicated in metastasis of gastric cancer [86]. SFRP1,
encoding secreted frizzled-related protein, a WNT signaling
pathway antagonist, is another ‘‘late’’ locus. SFRP1 was previously
examined in AAH lesions, and was found to be DNA methylated
in 11–14% of AAH lesions [35]. In our hands, DNA methylation
of SFRP1 in AAH was even less frequent, and we see little DNA
methylation in AIS. Like Licchesi et al., we observe dramatic
hypermethylation of SFRP1 in adenocarcinoma (Figure 5),
suggesting that the DNA methylation of this gene may be a key
change associated with invasion. Transcriptional silencing of
SFRP1 by DNA methylation and loss of heterozygosity in lung
cancer have been documented, supporting a role for this gene as a
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DNA Methylation in AAH AIS and Lung Adenocarcinoma
tumor suppressor [87,88], and SFRP1 hypermethylation was found
to be associated with lymph node metastasis and progression [88].
The silencing of SFRP1 is especially of interest since the WNT
pathway was recently implicated in lung adenocarcinoma
metastasis [89]. TWIST1, encoding a helix-loop-helix transcription
factor, was identified as DNA methylated in lung cancer based on
a genome-wide screen for genes reactivated in lung cancer cell
lines by 5-aza-29deoxycitidine, a DNA methyltransferase inhibitor
[90]. The locus has also been found to be highly methylated in
metastatic breast cancer [91]. Intriguingly, overexpression of
TWIST1 has been linked to invasion and metastasis in
hepatocellular carcinoma and oesophageal cancer [92,93]. These
observations suggest that further studies of TWIST1 to clarify its
role in invasion and metastasis are warranted. Of the genes we
characterized as becoming DNA methylated as AIS becomes
invasive, CDX2 has been least well studied. In colorectal cancer,
its DNA methylation appears to cause silencing and seems to be
associated with advanced stage disease and poor prognosis
[94,95]. In the study of stage I lung cancer patients mentioned
above, DNA methylation of RASSF1, a ras-associated putative
tumor suppressor, was also found to be associated with
recurrence [58]. Numerous groups have reported RASSF1 DNA
methylation in lung cancer [37,96–98], and methylation of this
gene has been associated with poor prognosis [96,99] and later
stage cancer [100]. The latter observations would appear to be in
agreement with our characterization of RASSF1 DNA methylation as associated with the transition from in situ cancer to
invasive cancer. While we observed occasional hypermethylation
of RASSF1 in both AAH and AIS, the frequency in these
preinvasive lesions was low, and DNA methylation levels were
also low. The DNA methylation frequency we observed in the
tumors was comparable to that found by us and others [37,96–
98]. It is interesting therefore that RASSF1 DNA methylation has
been found in the sputum of smokers prior to the detection of
overt lung cancer [101]. In the only other analysis of RASSF1
DNA methylation in AAH lesions [34], methylation of the locus
was reported in almost 30% of AAH, a frequency that
approaches that reported for tumors. The lower frequency we
observe in AAH in our study might be attributable to our use of a
quantitative technique to measure DNA methylation, and to the
fact that our probe/primer set detects methylation of 6 CpGs in
the amplicon, thus providing a more strict measurement of
hypermethylation.
To obtain an indicator for the timing of hypomethylation with
respect to lung adenocarcinoma development, we used the mean
of two repeat-based probes used as measures for global DNA
methylation [39]. To our knowledge, global DNA methylation has
not been previously investigated in the putative preinvasive stages
of lung adenocarcinoma. We observe highly significant hypomethylation only in adenocarcinoma, suggesting that pervasive
global hypomethylation is a later event than hypermethylation.
However, it should be noted that there is quite a wide spread of
global DNA methylation levels in all of the sample types we tested
(Figure 6). A recent study of global hypomethylation in stage 1
lung cancer found it to be significantly associated with stage IB vs.
IA, larger tumors and less differentiated morphology [102],
indicating that it may indeed be a later rather than earlier event.
As a comparison for the studied (pre)malignant lesions, we
examined two types of histologically normal lung tissue, AdjNTL
and MetNTL. We observed no increased DNA methylation of the
15 loci in AdjNTL compared to MetNTL. This is especially
telling, since the median age of the MetNTL subjects was slightly
younger (Table S2), and increased DNA methylation with age has
been reported [103]; if observed, a slightly higher DNA
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methylation in AdjNTL could have been attributed to the small
age difference. The lack of higher DNA methylation in AdjNTL
strongly suggests that there is no field defect for these loci, at least
when compared to histologically normal lung from patients with a
metastasis to the lung. We did observe significantly higher DNA
methylation of PTPRN2 in MetNTL. One possible explanation is
that something is different about PTPRN2 in the cases from which
MetNTL was obtained. We have no basis for assuming that
PTPRN2 values for AdjNTL are not representative and for some
reason were abnormally low.
While we did not find increased DNA methylation in any of our
15 DNA hypermethylation loci between high-grade or low-grade
AAH, in an unsupervised analysis we identified a small group of
five AAH lesions that showed significantly higher levels of DNA
methylation in seven loci: 2C35, CDKN2A ex2, CDX2, HOXA1,
NEUROD1, TMEFF2 and TWIST1. Whether this elevated DNA is
somehow related to the propensity to progress will require further
studies, but it is notable that four of these loci are ones that were
designated ‘‘intermediate’’ for increased DNA methylation in AIS.
The four patients carrying the AAH that were more highly
methylated did not consistently show unusually high DNA
methylation in their other lesions, confirming that lesions found
in patients are independent. The small number of lesions that
clusters separately from the main group of AAH would be in
keeping with a model in which the majority of AAH lesions may
never progress. The five AAH lesions in the small cluster were a
mixture of HG and LG lesions, again indicating no link between
hypermethylation and grade designation in AAH. One could
wonder whether the 5 separately clustering AAH samples were the
ones driving the designation of CDKN2A ex2 as an ‘‘early’’
hypermethylation change, since they exhibited higher levels of
DNA methylation of this locus than other AAH samples.
However, when we reanalyzed the data set with the omission of
these five samples the difference in DNA methylation of CDKN2A
ex2 between AdjNTL and AAH was still highly significant
(p,0.000001), supporting its designation as an ‘‘early’’ DNA
methylation event occurring as hyperplasia develops in the
peripheral lung.
Of interest was the observation that the patient for whom two
AAH lesions partitioned to the small cluster had 7 AIS lesions.
Comparison of DNA hypermethylation levels between single AAH
or AIS lesions and those from subjects in whom two or more
lesions were found showed no statistical differences in PMR levels
for any of the CpG islands, and the distribution of PMR values
was comparable to that of the single AAHs or AISs (not shown).
Thus, it would not appear that persons with many AAH or AIS
lesions show generally increased DNA methylation levels in these
lesions.
For those loci for which it is unknown whether their DNA
methylation might contribute to cancer (such as 2C35), further
experiments will be required to determine whether hypermethylation has functional consequences. Examining the biological
consequences of sequential gene silencing, for example in AAHor AIS-derived cell lines [104], will help confirm the role of the
genes under study in lung adenocarcinoma development and
progression. Further delineating the nature and timing of
epigenetic hits, which are in principle reversible, is potentially
highly relevant for epigenetic therapy of early lung cancer, and
perhaps for cancer prevention. Lastly, irrespective of the biological
effects of hypermethylation at each locus, the presence of DNA
methylation characteristic of each type of lesion can be used to
inform the generation of biomarkers specific for the different
developmental stages of lung adenocarcinoma.
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Supporting Information
Acknowledgments
Table S1 Genes, primers and probes.
The authors thank Gyeong Hoon Kang for the design of the EYA4 probe/
primer set and the Laird-Offringa and Laird lab members for critical
comments and advice.
(DOC)
Table S2 Information on subjects from whom samples were
obtained.
(DOC)
Author Contributions
Conceived and designed the experiments: SAS JSG KMK IAL-O.
Performed the experiments: SAS JSG. Analyzed the data: SAS ADJ
KDS IAL-O. Contributed reagents/materials/analysis tools: MNF MC
KMK. Wrote the paper: SAS KDS KMK IAL-O.
Table S3 Comparison between high-grade and low-grade AAH
lesions.
(DOC)
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