Carcinogenesis vol.30 no.3 pp.
416–422, 2009
doi:10.1093/carcin/bgp006
Advance Access publication January 6, 2009
Epigenetic profiling reveals etiologically distinct patterns of DNA methylation in head
and neck squamous cell carcinoma
Carmen J.Marsit1,y,
, Brock C.Christensen1,y, E.Andres
Houseman2,3, Margaret R.Karagas4, Margaret
R.Wrensch5,6, Ru-Fang Yeh6, Heather H.Nelson7, Joseph
L.Wiemels5,6, Shichun Zheng5, Marshall R.Posner8,
Michael D.McClean9, John K.Wiencke5 and Karl
T.Kelsey1,3
1
Department of Pathology and Laboratory Medicine, Brown University, Box
G-E537, Providence, RI 02912, USA, 2Department of Biostatistics, Harvard
School of Public Health, Boston, MA 02115, USA, 3Department of
Community Health, Center for Environmental Health and Technology, Brown
University, Providence, RI 02912, USA, 4Department of Community and
Family Medicine, Dartmouth Medical School, Lebanon, NH 03766, USA,
5
Department of Neurological Surgery and 6Department of Epidemiology and
Biostatistics, University of California—San Francisco, San Francisco, CA
94143, USA, 7Division of Epidemiology and Community Health, Masonic
Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA,
8 HPV16, smoking, betel nut use and methylation of specific genes
Head and Neck Oncology Program, Dana-Farber Cancer Center, Boston, MA
02215, USA and 9Department of Environmental Health, Boston University
School of Public Health, Boston, MA 02118, USA


To whom correspondence should be addressed. Tel: þ1 401 863 6508;
Fax: þ1 401 863 9008;
Email: carmen_marsit@brown.edu
Head and neck squamous cell carcinomas (HNSCCs) represent
clinically and etiologically heterogeneous tumors affecting
>40 000 patients per year in the USA. Previous research has iden-
tified individual epigenetic alterations and, in some cases, the re-
lationship of these alterations with carcinogen exposure or patient
outcomes, suggesting that specific exposures give rise to specific
types of molecular alterations in HNSCCs. Here, we describe
how different etiologic factors are reflected in the molecular char-
acter and clinical outcome of these tumors. In a case series of
primary, incident HNSCC (n 5 68), we examined the DNA meth-
ylation profile of 1413 autosomal CpG loci in 773 genes, in relation
to exposures and etiologic factors. The overall pattern of epigenetic
alteration could significantly distinguish tumor from normal head
and neck epithelial tissues (P < 0.0001) more effectively than spe-
cific gene methylation events. Among tumors, there were significant
associations between specific DNA methylation profile classes and
tobacco smoking and alcohol exposures. Although there was a sig-
nificant association between methylation profile and tumor stage
(P < 0.01), we did not observe an association between these profiles
and overall patient survival after adjustment for stage; although
methylation of a number of specific loci falling in different cellular
pathways was associated with overall patient survival. We found
that the etiologic heterogeneity of HNSCC is reflected in specific
patterns of molecular epigenetic alterations within the tumors and
that the DNA methylation profiles may hold clinical promise
worthy of further study.
Introduction
Head and neck squamous cell carcinoma (HNSCC) is a physically,
etiologically and molecularly heterogeneous disease, with an annual
incidence in the USA of .40 000 cases. The majority of head and
neck cancers are associated with tobacco and alcohol use, acting both
Abbreviations: FFPE, formalin-fixed paraffin embedded; HNSCC, head and
neck squamous cell carcinoma; HPV, human papilloma virus; OOB, out of bag;
RF, random forest.
y
These authors contributed equally to this work.
Ó The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
independently and synergistically (1,2). However, human papilloma
virus (HPV), particularly the high-risk type 16, is associated with
20–25% of HNSCC, and individuals with HPV-positive disease com-
pared with HPV-negative have better overall survival (3,4). Given the
established association of etiologic factors with clinical outcome,
identifying the molecular character of tumors arising from varying
exposures will aid in understanding the mechanisms influencing prog-
nosis and provide novel targets for diagnosis and therapy of HNSCC.
Study of the contribution of epigenetic alterations to tumor biology
is now a vast field, and it is widely accepted that epigenetic alterations
in target tissues are part of the causal path to the development of
malignancy (5,6). DNA methylation-associated epigenetic silencing
of tumor suppressor genes is an aberrant mark of cancer with consid-
erable specificity. DNA hypermethylation in HNSCCs targets genes in
pathways such as DNA repair, cell cycle control, apoptosis, angiogen-
esis, cell–cell interaction and metastasis (7). Associations among
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have been identified (8–10). These findings, though, have focused
on single gene methylation alterations and their associations to ex-
posures, but have not examined how exposures might be influencing
the overall processes leading to epigenetic alteration. We have pre-
viously demonstrated that exposures and age, in bladder cancer, lead
to an increased propensity for gene promoter hypermethylation in
a panel of 16 tumor suppressor genes (11). Using now available
high-throughput technologies, we are better equipped to understand
the process by which carcinogenic exposures act to alter the DNA
methylation status of a developing tumor.
We aim to more completely understand the etiology of epigenetic
alterations by examining the relationships between these alterations
and carcinogen exposures. In this manner, we hope to define novel path-
ways through which HNSCC can arise and aid in the development of
diagnostic screening tools and targeted therapies. We characterized
DNA methylation profiles of primary human HNSCC tumors by exam-
ining DNA methylation status of 1400 CpG sites in 800 cancer-
related genes in a population-based case series of incident, primary
HNSCC and non-diseased head and neck epithelium. Both the diagnostic
and prognostic utility of these markers were defined; and uniquely, we
also revealed how etiologic factors responsible for head and neck carci-
nogenesis are associated with the molecular character of these tumors.
Subjects and methods
Study population
The study population has been described previously (8,12). Briefly, incident
cases of histologically confirmed HNSCC were identified from nine medical
facilities in the Boston, MA, metropolitan area. Diagnoses were confirmed by
an independent study pathologist. All cases enrolled in the study provided
written informed consent as approved by the Institutional Review Boards of
the participating institutions. Archived pathology specimens were used for
analysis of promoter hypermethylation, and a total of 42 formalin-fixed par-
affin-embedded (FFPE) and 26 fresh-frozen tumor samples were selected for
analysis. Data on HPV16 tumor DNA status and serology from the parent case–
control study (3) have been previously reported. Demographic and exposure
information was collected through self-administered questionnaires and clin-
ical information through medical chart reviews. Table I shows the demographic
characteristics of the final population studied. In addition to the case tumor
tissues, non-malignant head and neck tissues from individuals without head
and neck cancer were obtained from the National Disease Research Inter-
change. Clinicopathologic information is limited by this anonymous tissue
bank, but all samples were obtained from patients who were not previously
diagnosed with any cancer, and thus whose cause of death was not cancer
related.
DNA extraction and methylation analysis
For FFPE tissues, tumor sections with the greatest proportion of malignant
tissue were selected by the study pathology for use in our molecular analyses.
416
 Epigenetic profiling in HNSCC

Subsequent analyses were carried out in The R Package. For exploratory/

Table I. Characteristics of the subjects with tissue involved in methylation visualization purposes, hierarchical clustering using the Manhattan metric and
analysis average linkage was performed. Associations between sample type or cova-
riates such as age or gender and methylation at individual CpG loci were tested
Characteristic HNSCC cases Non-diseased with a generalized linear model. The beta distribution of average beta values
(n 5 68) head and neck was accounted for with a quasibinomial logit link with an estimated scale
tissues (n 5 11) parameter constraining the mean between 0 and 1. In contrast, array-wide
scanning for autosomal CpG loci (n 5 1413) associations with sample type
Age, mean (±SD) 57.6 (11.4) 66.2 (7.9)
or covariate used false discovery rate correction and q-values computed by the
Gender, n (%)
qvalue package in R. An false discovery rate q-value of ,0.05 was considered
Female 14 (21) 3 (27)
significant for this examination. Initial analyses were performed to examine if
Male 54 (79) 8 (73)
there were systematic differences in methylation profile between FFPE and
Sample location, n (%)
fresh tumor samples. Clustering did not reveal linkage by sample type, nor was
Oral 35 (52) 3 (28)
any association observed across individual loci, thus these sample types (FFPE
Pharyngeal 26 (38) 4 (36)
and fresh) were combined in all subsequent analyses.
Laryngeal 7 (10) 4 (36)
The R Package was also used to build classifiers of sample type with the
Cigarette smokinga, n (%)
random forest (RF) approach, a supervised classification analysis. RF is a tree-
Never 16 (24) —
based classification algorithm similar to Classification and Regression Tree
Former 38 (56) —
(14–16) and was performed on CpG average beta values using RF R package
Current 13 (20) —
version 4.5-18 by Liaw and Wiener. RF builds each individual tree by taking
Lifetime average packs 1.3 (0.5) —
a bootstrap sample (sampling with replacement) of the original data and on
per day, mean (±SD)a
average about one-third of the original data are not sampled [out of bag
Number of years smoking, 32.2 (14.5) —
(OOB)]. Those sampled are used as the training set to grow the trees, and
mean (±SD)a
the OOB data are used as the test set. At each node of the tree, a random
Lifetime pack-years smokeda 41.1 (26.1) —

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Lifetime average alcoholic 28.3 (35.5) —
drinks per week
Tumor HPV16 DNA statusb
Negative 56 (85%) —
Positive 10 (15%) —
a the OOB obtained on the data set with the null distribution of OOB errors
Smoking data not available on one case and metrics of smoking (average
packs per day, years smoked and pack-years smoked) based only on current
and former smokers.
b
HPV data not available on two cases.
Three 20 lM sections were cut from each FFPE tumor sample and the sections
were transferred into microcentrifuge tubes. The paraffin was dissolved using
Histochoice Clearing Agent (Sigma–Aldrich, St Louis, MO) followed by two
washes with 100% ethanol and one wash with phosphate-buffered saline. The
samples were then incubated in sodium dodecyl sulfate lysis solution (50 mM
Tris–HCl, pH 8.1, 10 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl
sulfate) with proteinase K (Qiagen, Valencia, CA) overnight at 55°C. De-
crosslinking was performed by adding NaCl (final concentration 0.7 M) and
incubating at 65°C for 4 h. DNA was recovered using the Wizard DNA clean-
up kit (Promega, Madison, WI) according to the manufacturer’s protocols. For
fresh-frozen tumor tissues and peripheral blood samples, DNA was extracted
using the QIAamp DNA mini kit according to the manufacturer’s protocol
(Qiagen). Sodium bisulfite modification of the DNA was performed using
the EZ DNA Methylation Kit (Zymo Research, Orange, CA) following the
manufacturer’s protocol, with the addition of a 5 min initial incubation at 95°C
prior to addition of the denaturation reagent. The de-crosslinking steps in the
extraction as well as the 95°C incubation ensure more complete melting of
the DNA and thus more complete sodium bisulfite conversion, especially for
the highly cross-linked formalin-fixed specimens. Illumina GoldenGateÒ
methylation bead arrays were used to simultaneously interrogate 1505 CpG
loci associated with 803 cancer-related genes. This is a commercially available
array designed by Illumina to interrogate genes with CpG islands considered
cancer related. Bead arrays were run at the University of California-San
Francisco Institute for Human Genetics, Genomics Core Facility according
to the manufacturer’s protocol and as described in ref. (13).
Statistical analysis
We assembled data with BeadStudio Methylation software from the array man-
ufacturer Illumina (San Diego, CA). All array data points are represented by
fluorescent signals from both methylated (Cy5) and unmethylated (Cy3) alleles,
and methylation level is given by b 5 [max (Cy5, 0)]/(jCy3j þ jCy5j þ 100),
the average methylation (b) value is derived from the 30 replicate methylation
measurements for each loci. At each locus for each sample, the detection P-value
is defined as 1  P-value computed from the background model characterizing
the chance that the signal was distinguishable from negative controls. Using this
as a metric for quality control for sample performance, seven samples (9%), all
FFPE, where ,75% of loci had a detection P-value ,1  105, were dropped
from analysis. A similar quality control for CpG loci eliminated those with
median detection P-value .0.05 (n 5 8, 0.5%).
sample of m out of the total M variables is chosen and the best split ispfound ffiffiffiffiffi
among the m variables. The default value for m in the pRF
ffiffiffiffiffi R package ispffiffiffiffiM
ffi . In
this analysis, we will test a range of m from half of M to two times M and
will use the m that gives the lowest prediction error. The OOB error rate is the
percentage of time the RF prediction is incorrect. A test for association be-
tween methylation (predictors) and sample type was conducted by comparing
obtained by permuting sample type labels and running the RF procedure
20 000 times.
For inference, data were clustered using a mixture model (17) with a mixture
of beta distributions (18), and the number of classes was determined by Bayes-
ian information criterion (19,20). The mixture model was fit by recursively
partitioning the data using a 2-class mixture model, with Bayesian information
criterion used as a criterion for the split, as described in ref. (21). Class mem-
bership was obtained from the mixture model and subsequent univariate asso-
ciations were tested via permutation test with 10 000 permutations each. For
continuous variables, the Kruskal–Wallis test statistic was used, whereas for
categorical variables, the standard chi-square goodness-of-fit test was used.
For those variables having a significant univariate association, multinomial lo-
gistic regression was used to model methylation class while controlling for
potential confounders. Because of the potentially large number of methylation
classes, logistic regression coefficients were regularized using a ridge (L2) pen-
alty, with coefficients for a common (non-intercept) covariate across outcome
levels shrunk toward zero (22,23) the tuning parameter was selected by minimiz-
ing Bayesian information criterion. To test multivariate associations with stage,
ordinary logistic regression and likelihood ratio tests were used. Cox proportional
hazards models and likelihood ratio tests of models with and without methylation
classes were used for survival analyses. In addition, we performed a locus-by-
locus analysis of the 500 most variable loci to examine associations with patient
survival using a Cox proportional hazards model, using false discovery rate cor-
rection and q-values computed by the qvalue package in R.
Pathway analysis was conducted with the use of Ingenuity Pathways Anal-
ysis (IngenuityÒ Systems, Redwood City, CA; www.ingenuity.com) (24). Ca-
nonical pathways analysis identified the pathways from the Ingenuity
Pathways Analysis library of canonical pathways that were most significant
to the data set. The top 500 most variable CpG gene loci tested locus-by-locus
for a survival association that was also associated with a canonical pathway in
the Ingenuity Pathways Knowledge Base were considered for the analysis.
Since we are using a cancer panel, the 500 CpG gene loci analyzed were also
used as the reference set to avoid bias toward cancer-related pathways by
selecting ‘user data set’ in the edit analysis parameters window. The significance
of the association between the data set and the canonical pathway was measured in
two ways: (i) a ratio of the number of gene loci from the data set that map to the
pathway divided by the total number of gene loci that map to the canonical
pathway is displayed and (ii) Fisher’s exact test was used to calculate a P-value
determining the probability that the association between the genes in the data set
and the canonical pathway is explained by chance alone.
Results
Characterization of the profile of DNA methylation alterations in non-
malignant head and neck epithelial tissues compared with HNSCC

417
C.J.Marsit et al.

tumor samples was completed using the Illumina Goldengate Meth-
ylation BeadArray. Unsupervised hierarchical clustering of the DNA
methylation data with Manhattan distance and average linkage as the
metric across the 1250 most variable autosomal loci (Figure 1A)
depicts relatively tight clustering of the non-malignant tissues com-
pared with the tumors, as well as the extent of variability in the
methylation beta value across the loci. In a locus-by-locus analysis
applying an false discovery rate cutoff q-value of 0.05, we identified
261 loci with significantly differential methylation between tumors
and normal (see supplementary Figure S1 available at Carcinogenesis
Online). Of those, 125 loci showed greater methylation in tumors
compared with normal, whereas 136 loci exhibited lower methylation
levels in tumors compared with normal tissues (see list of loci in
supplementary Materials and Table 2, is available at Carcinogenesis
Online). The confusion matrix (Table II) resulting from RF analysis
shows which samples are correctly classified, those that are

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Fig. 1. (A) Unsupervised hierarchical clustering and heat map of methylation beta values for 1250 most variable loci across all samples. (B) Recursive
partitioning mixture model classification of normal and tumor head and neck tissues using all methylation beta values resulting in eight classes whose average
methylation beta values are represented in the heat map. Distribution of normal and tumor samples within each class is depicted in pie charts on the right.

418
 Epigenetic profiling in HNSCC

misclassified and the misclassification error rate for each sample type. A recursive partitioning mixture model applied to methylation data
Five normal tissues (45%) were misclassified as tumor tissues, while from all autosomal loci in tumors and non-tumor head and neck
only two tumors were misclassified as normal tissues (3.0%). This epithelial tissues delineated 11 distinct methylation classes (Figure
results in an overall error rate of 8.86%, a significant improvement in 1B). This model demonstrates that methylation class membership
sample classification compared with the expected error under the null was a highly significant predictor of tumor status (permutation
hypothesis (P , 0.0001). These results, consistent with our previous P , 0.0001).
work, suggest that use of overall patterns of methylation alterations To examine how known risk factors for HNSCC are associated with
may have more utility in capturing the tumorigenic process than do these profiles, we utilized a case series approach and reconstructed
individual alterations. the recursive partitioning mixture models using only the tumor data
(Figure 2A), resulting in the delineation of six tumor-specific classes.
A permutation test with tumor stage, dichotomized as high (Stage III
Table II. RF classification of HNSCC tumor status using DNA methylation or IV) versus low (Stage I or II) revealed a significant association
between methylation class membership and stage (P , 0.01). A lo-
Tumor Non-diseased Error rate gistic regression model of stage (see supplementary Table 1 available
sample sample (%) at Carcinogenesis Online) suggested that inclusion of methylation
Tumor sample 66 2 2.9 class is significant in predicting stage (likelihood ratio P , 0.01)
Non-diseased sample 5 6 45 and that membership in Class 6 was associated with a significantly
reduced risk of high-stage disease (odds ratio 0.1, 95% confidence
Overall out of the bag (OOB) estimate of error rate 5 8.86%, permutation test interval 0.01, 1.0). Membership in Class 2 showed a similar protective
for association: P , 0.0001. effect, whereas membership in Class 5 was associated with an

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Fig. 2. Recursive partitioning mixture model classification of HNSCCs (A) resulting in six classes with average methylation beta values across loci depicted in
the heat map. (B) Average age of (C) lifetime average packs of cigarettes smoked per day by, (D) distribution of tumor location of and (E) lifetime average
alcoholic drinks per week consumed by patients whose samples are members of the distinct methylation classes depicted in (A).

419
C.J.Marsit et al.
increased risk of high-stage disease, although the small numbers of
tumors in these classes made these estimates unstable.
In order to identify if exposures leading to this disease are associ-
ated with these methylation classes, we examined the associations
between individual risk factors for HNSCC and these classes. Meth-
ylation class was significantly associated with patient age as a contin-
uous variable (permutation test P , 0.01, Figure 2B); methylation
Class 2 members had lower patient age, and Class 4 higher age com-
pared with other classes. Smoking intensity (packs per day) also sig-
nificantly differed across methylation classes (P , 0.04, Figure 2C);
Class 1 demonstrated lower smoking intensity, and 3 relatively high
intensity. However, we did not observe a significant association of
methylation class with smoking duration (years smoked) or pack-
years smoked. A borderline significant association was observed with
tumor site by methylation class (oral, pharyngeal and laryngeal,
P , 0.1) (Figure 2D). Tumor HPV16 DNA status also demonstrated
an association with methylation class that approached statistical sig-
nificance (P , 0.1, Figure 2E), patients in Class 4 had a greater prev-
alence of HPV-positive tumors. Finally, lifetime average drinks per
week also showed a strong differential trend by methylation class
(P , 0.1, Figure 2F).
Multinomial logistic regression results are shown in Table III, with
the classes numbered as they were in Figure 2A and with Class 5
serving as the referent class as this class had the largest membership.
The overall Wald P-value indicates whether the covariate significantly
differentiates class membership overall. Individual confidence inter-
vals for each covariate within a class identify the magnitude of any
association and significance of the association of a covariate on mem-
bership in that class compared with the referent class (Class 5), con-
ditional on membership in either class.
Patient age and average alcohol drinks per week each significantly
differentiated membership across classes (Wald P , 0.0001). Laryn-
geal tumors were less probably to be members of Class 1, and the odds
of membership in Class 1 were significantly reduced with each year of
age. In addition, the odds of membership in Class 1 compared with
Class 5 were significantly decreased by almost 20% for each addi-
tional pack of cigarettes smoked per day on lifetime average. Each
year of age reduced the odds of membership in Class 2 compared with
Class 5 by .10%, and tumors in this class were mostly likely to be
oral tumors compared with pharyngeal or laryngeal. Only age dem-
onstrated a significant effect on membership in Class 3 and Class 4,
leading to a reduced odds of membership in Class 3 compared with
Class 5, but an increased odds of membership in Class 4 compared
with Class 5. Class 6 tumors were significantly less probably to be
HPV-positive tumors, but more probably to be from patients with
Table III. Multinomial logistic regression of methylation class membership by etiologic factors
Covariate Class 1 OR Class 2 OR (95% CI),
(95% CI), n53
n 5 17
Age, per year 0.94 (0.94, 0.95) 0.89 (0.87, 0.91)
Tumor site
Oral Referent Referent
Pharyngeal 1.07 (0.93, 1.22) 0.95 (0.92, 0.98)
Laryngeal 0.95 (0.90, 1.00) 0.98 (0.97, 1.00)
Tumor HPV16 DNA status
Negative Referent Referent
Positive 1.00 (0.90, 1.11) 1.01 (0.95, 1.07)
Lifetime average packs 0.84 (0.71, 0.99) 0.93 (0.85, 1.01)
of cigarettes per
day smoked
Lifetime average 0.99 (0.99, 1.00) 1.00 (0.99, 1.01)
alcoholic drinks
per week
The odds ratio for each covariate in each class is conditional on membership in the given class compared with Class 5, the referent class (n 5 23). The model is
controlled for all covariates listed in the table. Results in bold italics are considered statistically significant (P , 0.05). OR, odds ratio; CI, confidence interval.
420
greater lifetime alcohol exposures. These results overall suggest that
differing etiologies of this disease influence the pattern of epigenetic
alteration observed in the resulting tumors.
Next, we examined if the DNA methylation profiles or methylation
at specific loci were associated with patient survival. Among the 68
samples examined for methylation using the array, there were 22
deaths and a mean of 2.75 years of follow-up among surviving
patients (range 0.75–5 years). We found no significant association
between methylation classes derived from the recursive partitioning
mixture modeling procedure among tumors and overall patient
survival, controlling for tumor stage and patient age.
Finally, we tested the hypothesis that biologic pathways, rather than
overall profiles of methylation, are important in determining survival.
To examine this hypothesis, we utilized Ingenuity Pathway Analysis
to examine which specific pathways were overrepresented among the
top 500 loci having both positive and negative correlation with sur-
vival as determined by loci-specific Cox proportional hazards analysis
(24). The pathways identified to be significantly overrepresented are
listed in Table IV, as well as correlation between the increase in
methylation beta value of the genes represented by that pathways
and patient survival, such that those with a positive correlation would
represent loci whose increasing methylation level is associated with
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improved patient survival (i.e. hazard ratio ,1), whereas those with
a negative correlation are loci where increasing methylation is asso-
ciated with poorer survival or a risk hazard ratio (.1). We also iden-
tified 18 loci with a false discovery rate ,20% in their association
with overall patient survival in models stratified by tumor stage and
controlled for patient age, and those loci are shown in supplementary
Table 2 (available at Carcinogenesis Online). Of note, only two
of these 18 loci (ZAP70 and GP1BB) are associated with a hazard
ratio .1, whereas 16 demonstrate a protective hazard ratio ,1. Such
a negative association with risk could indicate, in fact, that loss of
methylation at these loci may be associated with increased risk, as one
might expect from oncogene activation.
Discussion
This study revealed that patterns of epigenetic alteration, rather than
the identification of single or small numbers of epigenetic alterations
affecting individual genes, may hold greater utility in identifying the
process through which carcinogens act epigenetically to drive tumor-
igenesis as well as in providing useful diagnostic value to these mo-
lecular markers. The utility of profiles is probably due to the strong,
yet multidirectional, correlation between epigenetic alterations across
loci, as we have previously reported (25), and as clearly seen in the
Class 3 OR (95% CI), Class 4 OR Class 6 OR Overall
n54 (95% CI), (95% CI), Wald P
n54 n 5 17
0.97 (0.97, 0.98) 1.08 (1.04, 1.12) 0.98 (0.98, 0.98) 0.0001
0.001
Referent Referent Referent
0.98 (0.90, 1.06) 0.94 (0.88, 1.01) 0.90 (0.79, 1.01)
1.06 (0.95, 1.19) 0.98 (0.96, 1.01) 1.01 (0.92, 1.12)
0.32
Referent Referent Referent
0.98 (0.94, 1.01) 1.09 (0.98, 1.20) 0.93 (0.86, 1.00)
1.07 (0.94, 1.22) 0.95 (0.85, 1.06) 0.92 (0.81, 1.04) 0.07
1.01 (0.99, 1.02) 1.01 (0.99, 1.02) 1.02 (1.01, 1.02) 0.0001
 Epigenetic profiling in HNSCC

Table IV. Pathways significantly overrepresented in analysis of loci-specific associations with patient survival

Pathway Direction of correlation P-value Genes on array in pathway

with survival

Ephrin receptor signaling þ 0.009 SRC, EGF, CRK, EPHA2, GNG7, PDGFB, FGF1
SAPK/c-jun N-terminal kinase signaling þ 0.012 LCK, GADD45A, CRK, GNG7
Platelet-derived growth factor signaling þ 0.020 SRC, PDGFRA, CRK, JAK3, PDGFB
Cell cycle: G2/M DNA damage checkpoint regula- þ 0.030 CDKN2A, GADD45A, CDKN1A, SFN
tion
NF-jB signaling þ 0.039 LCK, BMP4, IL1RN, LTA, ZAP70, PDGFRA, EGF,
IRAK3
p53 signaling þ 0.040 CDKN2A, GADD45A, CDKN1A, SERPINB5, BAX,
SFN
Acute phase response signaling þ 0.043 IL1RN, IL6, RBP1
Hepatic fibrosis/hepatic stellate cell activation þ 0.045 IFNG, IFNGR2, EGF, MYH11, MMP2, BAX, IL6,
PDGFB, FGF1, COL1A1, CYP2E1, PDGFRA,
TGFB3
Synaptic long-term depression  0.045 IGF1, GUCY2D, PLA2G2A, NOS2A, NOS3, NPR2
Purine metabolism  0.049 TJP2, GUCY2D, PDE1B, NPR2

SAPK, stress activated protein kinase.

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present data. Our method for identification of methylation classes
based solely on the patterns of DNA methylation alterations derived
from the Goldengate Methylation BeadArray limits the bias intro-
duced by selection of candidate genes for examination and allows
for statistical inference, so that the association between these classes
and various clinical or risk factors can be assessed with rigor. It is also
critically important to note that it is not only hypermethylation of
specific loci that is used in defining these signatures of epigenetic
alterations between non-malignant and malignant tissues but also
hypomethylation of loci at particular CpG sites. The locus-by-locus
approach suggested that 125 loci have greater methylation in tumors
compared with normal, whereas 136 loci exhibit lower methylation
levels in tumors compared with normal tissues. The RF classification
suggests that using DNA methylation at all CpG loci may be clinically
useful in confirming tumor, as misclassification of tumor as non-
tumor tissue occurred at a frequency of only 3%, but that as a screening
tool, differentiating non-tumor from tumor tissue, misclassification
may be higher. The recursive partitioning approach defined patterns
that can well differentiate between non-diseased oral epithelium and
malignant tissues, with class membership highly significantly associ-
ated with tissue type (P , 0.0001). Similarly, looking specifically
within tumors, these methylation alterations patterns significantly dif-
ferentiated between low-stage disease (I, II) and high (III, IV)-stage
disease (P , 0.01) with Class 6 being more probably of lower stage
than Class 1, controlling for the other methylation patterns. It should
be noted that differentiation of adjacent normal tissue in patients with
tumors may be more complicated than differentiation of tumor from
normal tissue of completely non-diseased individuals, and such an
examination is worthy of further study.
Patients whose tumors comprised Class 3 had greater exposures to
carcinogens causal to this disease, namely tobacco smoke and alco-
hol, and their tumors were more often laryngeal (Table III). Thus, the
chemical carcinogens, particularly those in cigarette smoke, may be
leading to this specific pattern of epigenetic alteration, either directly
or through mutation of key genes related to the control of DNA
methylation. On the other hand, the patients whose tumors comprised
Class 1, demonstrated modest or low exposures to tobacco and alco-
hol, were not, for the most part, HPV16 DNA positive within the
tumors and were relatively young compared with classes 3–6. Thus,
these individuals may represent a group of people with true suscepti-
bility for this disease, such that even low to modest exposure can
initiate oral carcinogenesis. Further studies are necessary to confirm
these observations in a larger series of tumors and may also consider
integration of these profiles with genome-wide polymorphism studies
to aid in identifying the genes or pathways responsible for this in-
creased susceptibility and thereby aid in the prevention or treatment of
this disease.
The individuals whose tumors reside in classes 4–6 demonstrated
a greater intensity of tobacco smoke exposure as well as an elevated
exposure to alcohol and appeared to be older than individuals of the
other classes. Thus, the methylation patterns exhibited by these indi-
viduals may be driven more by these exposures and indicate that they
are somewhat less susceptible to the disease, as they presented at
a later age and required greater exposure to attain this disease.
We were unable to observe any significant associations between
these methylation profiles and overall patient survival. This may in-
dicate that our approach (defining methylation classes) classifies only
the phenotypic results of methylation and is not integrating the obvi-
ously crucial results of genetic changes. Indeed, the epigenetic alter-
ation profile may be driven predominantly by exposures that do not
relate to patient outcome. Our analyses of pathways overrepresented
among the loci demonstrating a relationship to patient survival suggests
that patient outcome is more probably determined not by the pattern of
methylation but by the combined targets of genetic and epigenetic
alteration. The most significantly overrepresented pathway, in this anal-
ysis, was the ‘ephrin receptor-signaling pathway’, containing a number
of oncogenes such as SRC, EGF and EPHA2. The results of the Cox
model demonstrate a positive relationship between methylation beta
value of these loci and improved patient survival, or more appropriately
that loss of methylation at these loci is associated with poorer patient
survival. Overexpression of EPHA2, for example, has been linked to
invasiveness in breast (26) and ovarian (27) tumors, and in general,
deregulation and overexpression of this family of genes are related to
changes in cell motility, morphology, migration and adhesion—all
marks of an aggressive phenotype (28,29). Similarly, loss of methyla-
tion of the stress activated protein kinase (SAPK)/c-jun N-terminal
kinase and platelet-derived growth factor-signaling pathways,
traditional oncogenic growth-signaling pathways, probably represents
increasing tumor growth leading to poorer patient outcome. Gene-
specific analyses also suggest that loss of methylation of critical onco-
genic growth factors and receptors including HGF, FGF, ATP10A and
NTRK3 is associated with poorer patient survival, whereas hyperme-
thylation of ZAP70 and GP1BB is also associated with poor survival,
further suggesting that these genes may be acting as novel tumor
suppressors in HNSCC. Additional analyses using a larger independent
set of tumors will aid in confirming these results and determining if
a targeted panel of genes, such as those predicted by this array-based
approach, can be clinically useful as prognostic indicators in this
disease. In addition, these data suggest that integrative analyses of
human tumor samples, including epigenetic alteration, genetic copy
number changes and mutation, may be necessary to truly understand
the biology and better identify meaningful disease biomarkers.
HNSCC is a relatively common tumor, characterized by its aggres-
siveness and high morbidity, particularly at later stages, as well as for

421
C.J.Marsit et al.
the distinct clinical outcomes associated with its disparate etiologies.
Molecular characterizations of HNSCC tumors have yet to clearly
identify pathways responsible for driving the differential aggressive-
ness or treatment response related to HPV etiology. Due to its aggres-
siveness at high stages, the diagnostic and prognostic utility of somatic
molecular alterations including DNA methylation and genetic altera-
tions in accessible materials, such as saliva or buccal swabs, at early
stages of disease development is highly desirable, but has proven elu-
sive due to a general lack of markers with appropriate levels of sensi-
tivity and specificity (30). Identification of novel sites or patterns of
DNA methylation alterations in tumors may provide the necessary
sensitivity and specificity to eventually make non-invasive, early di-
agnosis of this disease possible. In addition, understanding how the
disease risk factors drive these alterations may allow for more specific
diagnostic panels, as well as more targeted preventative strategies based
on the molecular character of the resulting disease. It is clear that
exposures causal to this disease not only can influence the genetic
make-up of the tumor has been previously demonstrated but also sig-
nificantly and distinctly alter the epigenetic profile. This concept that
carcinogens and lifestyle factors contribute to tumorigenesis through
epigenetic mechanisms is critical and holds great promise in disease
prevention and treatment. In order to define those individuals at greatest
risk and to limit the effect of carcinogens in these individuals, the
epigenetic character of the individual and the epigenetic mechanism
of the carcinogens must be considered. In addition, treatment aimed at
the epigenome holds great promise as we continue to understand the
complex and powerful contribution, this mode of somatic alteration
plays in tumorigenesis.
Supplementary materials
Supplementary Tables 1 and 2 and Figure S1 can be found at http://
carcin.oxfordjournals.org/
Funding
Flight Attendants Medical Research Institute to C.J.M. and M.D.M.;
National Institutes of Health (R01CA078609, R01CA100679,
R01CA52689, P50CA097257); Tendrich/Berkow Fund; Friends of
the Dana-Farber Cancer Institute.
Acknowledgements
Conflict of Interest Statement: None declared.
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Received October 8, 2008; revised November 24, 2008;
accepted December 22, 2008