Published OnlineFirst May 29, 2019; DOI: 10.1158/1078-0432.
CCR-18-3720
Translational Cancer Mechanisms and Therapy
Molecular Classification of Epithelial Ovarian
Cancer Based on Methylation Profiling: Evidence
for Survival Heterogeneity
Clara Bodelon1, J. Keith Killian2, Joshua N. Sampson1, William F. Anderson1,
Rayna Matsuno3,4, Louise A. Brinton1, Jolanta Lissowska5, Michael S. Anglesio6,7,
David D.L. Bowtell8,9, Jennifer A. Doherty10, Susan J. Ramus8,11, Aline Talhouk6,
Mark E. Sherman1,12, and Nicolas Wentzensen1
Abstract
Purpose: Ovarian cancer is a heterogeneous disease that can
be divided into multiple subtypes with variable etiology,
pathogenesis, and prognosis. We analyzed DNA methylation
profiling data to identify biologic subgroups of ovarian cancer
and study their relationship with histologic subtypes, copy
number variation, RNA expression data, and outcomes.
Experimental Design: A total of 162 paraffin-embedded
ovarian epithelial tumor tissues, including the five major
epithelial ovarian tumor subtypes (high- and low-grade
serous, endometrioid, mucinous, and clear cell) and
tumors of low malignant potential were selected from two
different sources: The Polish Ovarian Cancer study, and
the Surveillance, Epidemiology, and End Results Residual
Tissue Repository (SEER RTR). Analyses were restricted to
Caucasian women. Methylation profiling was conducted
using the Illumina 450K methylation array. For 45 tumors
array copy number data were available. NanoString gene
Introduction
Ovarian cancer is the fifth leading cause of cancer-related death
among women in the United States (1). Currently, there is no
1
Division of Cancer Epidemiology and Genetics, NCI, NIH, Bethesda, Maryland.
2
Center for Cancer Research (CCR), NCI, NIH, Bethesda, Maryland. 3Foundation
Medicine Inc., Cambridge, Massachusetts. 4University of California, San Diego,
California. 5M. Sklodowska-Curie Memorial Cancer Center and Institute of
Oncology, Warsaw, Poland. 6Department of Pathology and Laboratory Medi-
cine, University of British Columbia, Vancouver, Canada. 7Department of Obstet-
rics and Gynaecology, University of British Columbia, Vancouver, Canada.
8
The Kinghorn Cancer Center, Garvan Institute of Medical Research, Sydney,
Australia. 9Peter MacCallum Cancer Center, Melbourne, Australia. 10Department
of Population Health Sciences, Huntsman Cancer Institute, University of Utah,
Salt Lake City, Utah. 11School of Women's and Children's Health, University of
New South Wales, Sydney, Australia. 12Mayo Clinic, Jacksonville, Florida.
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Clara Bodelon, NCI, 9609 Medical Center Drive,
Bethesda, MD 20892. Phone: 240-276-7327; Fax: 240-276-7838; E-mail:
clara.bodelon@nih.gov
Clin Cancer Res 2019;XX:XX–XX
doi: 10.1158/1078-0432.CCR-18-3720

2019 American Association for Cancer Research.
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Clinical
Cancer
Research
expression data for 39 genes were available for 61 high-
grade serous carcinomas (HGSC).
Results: Consensus nonnegative matrix factorization clus-
tering of the 1,000 most variable CpG sites showed four major
clusters among all epithelial ovarian cancers. We observed
statistically significant differences in survival (log-rank test, P
¼ 9.1  107) and genomic instability across these clusters.
Within HGSC, clustering showed three subgroups with sur-
vival differences (log-rank test, P ¼ 0.002). Comparing models
with and without methylation subgroups in addition to pre-
viously identified gene expression subtypes suggested that the
methylation subgroups added significant survival information
(P ¼ 0.007).
Conclusions: DNA methylation profiling of ovarian cancer
identified novel molecular subgroups that had significant
survival difference and provided insights into the molecular
underpinnings of ovarian cancer.
effective screening strategy, and most cancers are detected at
advanced stages limiting curative therapies. Ovarian cancer is a
histologically heterogeneous set of diseases with approximately
90% of cases being of epithelial origin. The major histotypes of
malignant epithelial ovarian cancers (EOC) include serous (high-
and low-grade), endometrioid, clear cell, and mucinous (2).
Serous tumors account for 60%–70% of EOC, endometrioid
approximately 15%, clear cells for 5%, and mucinous for approx-
imately 10% (3–7), although these rates vary globally and have
changed over time (8). Of all these, high-grade serous carcinomas
(HGSC) represent the most common histotype and are associated
with poor prognosis (3, 5). Ovarian tumors of low malignant
potential (LMP) or borderline tumors account for 15% of all
epithelial neoplasms, and are generally considered apart from
extraovarian metastasis because of differences in proposed path-
ogenesis and highly favorable clinical outcomes in the absence of
invasive implants (9, 10).
Several studies have shown that the major histologic
subtypes of EOC have different risk factors (11), genetic
susceptibilities (12–14), and clinical response to therapy (15, 16).
These histotypes also differ at the molecular level. Serous tumors,
mostly high grade, were the only histologic subtypes included
in The Cancer Genome Atlas (TCGA; ref. 17). Results from this
large-scale genomics analysis showed that these tumors have
near ubiquitous TP53 mutations (96% of tumors) and a high
OF1
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Bodelon et al.
Translational Relevance
Ovarian cancer is a heterogeneous disease with several
histologic subtypes with different etiology, pathogenesis, and
prognosis. Identifying key molecular characteristics that differ
across these subtypes could refine tumor classification beyond
histology alone and inform about potential treatments and
etiology, and help in the development of new approaches for
prevention and early detection. In this analysis of epithelial
ovarian tumor DNA methylation, that included high- and low-
grade serous, endometrioid, mucinous, and clear cell, and
tumors of low malignant potential, we showed that there are
four methylation-defined subgroups of epithelial ovarian can-
cer with survival differences, similar to histologic subtypes.
Within high-grade serous carcinomas, we found methylation
subgroups that added significant survival information to pre-
viously identified gene expression subtypes.
level of copy number alterations. TCGA analyses confirmed four
transcriptional subtypes that were previously described for
HGSC (18). Large-scale integrated characterization of the other
subtypes is mostly lacking. Mutations in BRAF, KRAS, PTEN, and
B-catenin have been shown in endometrioid, clear cell, low-grade
serous, mucinous EOC (19).
There is increasing evidence that the different histotypes of
EOC have different cells of origin, including that many HGSCs
originate from secretory cells in the fimbria of the fallopian
tubes, while endometrioid and clear cell carcinomas appear to
arise from endometriosis, which itself may represent explanted
menstrual endometrium (20). The origin of mucinous tumors
is less well understood. It has been suggested that a possible
origin is in epithelial nests located in the tubo-peritoneal
junction (19). Of note, historically, many tumors classified as
ovarian mucinous carcinomas represent metastases to the ova-
ry, often from an occult gastrointestinal primary (21). Large-
scale identification of molecular features in tumors may help to
identify key molecular characteristics that differ across histo-
types, refine tumor classification beyond histology alone, and
inform about potential treatments and etiology, which could
lead to development of new approaches for prevention and
early detection of EOC.
DNA methylation is a key epigenetic regulator of gene expres-
sion across a variety of cellular processes (22). There is growing
interest in the role of DNA methylation in carcinogenesis and in
defining molecular subtypes that can assist in elucidating the
etiology, clinical characteristics, and outcomes of EOC. Genome-
wide methylation studies have focused mostly on a single sub-
type (14, 17, 23) with different studies using different technol-
ogies, experimental conditions, and analytical techniques, which
makes it difficult to compare results across the different histo-
types. Therefore, a more comprehensive study involving epigen-
ome-wide assessment of DNA methylation is needed to better
understand methylation status across different EOCs.
In two population-based studies, we analyzed DNA methyla-
tion in the major histologies of ovarian cancer to define subtypes
of EOC and compare these subtypes with the degree of chromo-
somal copy number alterations. Furthermore, we compared DNA
methylation within HGSC to previously identified subgroups
based on gene expression.
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Materials and Methods
Study population
Subjects included in this analysis were selected from two
studies, the Polish Ovarian Cancer Study (POCS; ref. 24) and
the NCI's Surveillance Epidemiology and End Results (SEER)
Residual Tissue Repository (RTR) study (25). Briefly, POCS was
a population-based case–control study that included eligible
cases diagnosed between June 1, 2001 and December 30, 2003,
residents in Warsaw or Lodz, Poland, and were aged 20–74 at
the time of diagnosis. Incident ovarian cancer cases were
ascertained through a rapid identification system coordinated
by five participating Polish hospitals, which cover approxi-
mately 85% of all cases diagnosed in the two cities (24). In
addition, cancer registries in Warsaw and Lodz were used to
identify cases missed by the rapid identification system. Med-
ical records were reviewed for pathology, treatment, and out-
comes information. An experienced gynecologic pathologist
(M. Sherman) reviewed the hematoxylin and eosin (H&E)
slides to determine the pathologic diagnosis used for this
analysis. Formalin-fixed paraffin-embedded (FFPE) tissue
blocks were available for a subset of ovarian cancer cases
(55%). Between two and four 1-mm cores were obtained
from these patients with cancer. The POCS was reviewed
and approved by the institutional review boards (IRB) of the
U.S. NCI, the M. Sklodowska Curie Institute of Oncology
and Cancer Center in Warsaw, and the Institute of Occupa-
tional Medicine in Lodz. This study is covered by Single
Project Assurances (SPA) in Warsaw (S-009741-04) and
Lodz (S-017191-01). All participants provided written
informed consent for use of clinical data and archival tissue
specimens. FFPE tumor blocks from 32 patients that did not
receive chemotherapy were included in the current analysis
(Supplementary Table S1).
The SEER RTR study included FFPE tissue blocks from pri-
mary tumors in the Hawaii and Iowa Tumor Registries (25).
Ovarian cancer cases in the Hawaii Tumor Registry were diag-
nosed from 1983 through 2004, which represented 38% of all
ovarian tumors in the Hawaii catchment area during that
period. Ovarian cancer cases in the Iowa Tumor Registry were
diagnosed from 1987 through 2003, representing 4% of all
ovarian tumors in the Iowa catchment area during that period.
Tumors were restricted to those from European ancestry wom-
en. Other demographic characteristics available, in addition to
race, were age and year at diagnosis and year of death. Tumor
characteristics included histology and stage from the American
Joint Committee on Cancer, and grade (25). Because SEER's
RTR data were anonymized, the NIH's Office of Human Sub-
jects Research (OHSR) designated the project as exempt from
IRB approval (OHSR #4081); nonetheless, IRB approvals were
provided at the Universities of Hawaii and Iowa. We retrieved
the available FFPE tissue blocks for primary invasive ovarian
carcinomas in the Hawaii and Iowa Discard Tumor Registries.
Tumor blocks from 144 Caucasian patients from the SEER RTRs
[60 (42%) from the Hawaii Tumor Registry and 84 (58%) from
the Iowa Tumor Registry] were included in this analysis (Sup-
plementary Table S1). H&E slides from all tumors went through
expert pathology reviewed by MES and the reviewed diagnosis
was used in this analysis. Tumors included in the analysis did
not receive neoadjuvant chemotherapy prior to their collection
based on review of pathology reports. Mortality data were
coded by year.
Clinical Cancer Research
 Published OnlineFirst May 29, 2019; DOI: 10.1158/1078-0432.CCR-18-3720
Methylation array
DNA extraction and genome-wide DNA methylation assays
using the Infinium HumanMethylation450 BeadChips (Illu-
mina) were performed as described previously (26, 27). Briefly,
FFPE tissue cores were deparaffinized using mineral oil and lysed
in Qiagen ATL buffer/proteinase K. NanoDrop UV absorbance
and PicoGreen dsDNA fluorometry (Invitrogen) were used to
measures DNA levels in the lysates. Fifty microliters of isolated,
purified DNA was sodium bisulfite–treated following the Zymo
Research EZ-96 Methylation Gold kit. Converted DNA was further
treated with the Illumina FFPE Restoration solution and 250 ng of
restored DNA was then run on the Infinium HumanMethyla-
tion450 BeadChip according to the standard protocol. Raw data
were extracted using the Genome Studio v2011.1.
Four samples were run in triplicate and one cell line (GIST 28T)
was included in quadruplicate across the two methylation plates
for quality controls. The average correlation across these replicates
was 0.984. A plot showing the intraclass-correlation coefficient
against the variance of the beta values is shown in the Supple-
mentary Fig. S1. Fourteen samples were excluded on the basis of
the lack of a bimodal distribution of the beta values, as expected
for this array. All these excluded samples were from the SEER
Hawaii Tumor Registry collected prior to 1993. Raw intensity data
were imported into the R programming software using the minfi
Bioconductor package (28). Data were preprocessed for back-
ground subtraction and controls normalization using the prepro-
cess Illumina function. Subset-quantile within array normaliza-
tion (SWAN) was performed to correct for type I and II probe
bias (29). Probes that were cross-reactive or located at single-
nucleotide polymorphisms were excluded (30). Probes that had a
detection P > 0.01, which indicated that they were indistinguish-
able from the background, were also excluded. Analyses were
restricted to probes in the 22 autosomes. A total of 362,498 CpG
sites remained for analysis.
Array copy number assay
A subset of invasive tumors for which enough DNA was
extracted that was not required for the methylation arrays (N ¼
47) were analyzed for chromosomal copy number alterations
using a commercial 180 K-feature array comparative genomic
hybridization (aCGH) assay (Agilent Inc.). We oversampled rare
subtypes (clear cell, mucinous, and endometrioid) for this anal-
ysis (see Supplementary Tables S1 and S2). Briefly, purified DNA,
prior to bisulfite conversion, was labeled with Cy5 and Promega
Male reference DNA was labeled with Cy3 using the Agilent
Genomic DNA ULS Labeling Kit with subsequent purification of
DNA on Kreatech genomic purification columns to remove excess
Cy3/Cy5. Equal amounts of Cy5-tumor DNA and Cy3-reference
DNA were combined and hybridized to Agilent 4  180K CGH
arrays for at least 40 hours. Slides were washed and scanned in the
Agilent Scanner C. Image data are extracted using the Agilent
Feature Extraction software and visual inspection of results is
performed using the Nexus 6.0 (Biodiscovery) software.
Background correction and normalization of the aCGH raw
signal intensities were done using the Bioconductor limma pack-
age with the minimum method for background correction and the
print-tip loess normalization, which takes into account signal
intensity and spatial position on the array (31). Diagnostic plots
were used to assure that signal biases were not present in the data
after normalization. Two samples were excluded on the basis of
this analysis, leaving data on 45 samples for analysis. Binary
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Methylation and Ovarian Cancer Survival
logarithm ratios (log2 ratios) of the two intensity channels were
then computed. For the subject with duplicate runs, the log2 ratios
of the two runs were averaged for analysis. The log2 ratios were
subjected to the circular binary segmentation (CBS) algo-
rithm (32) by means of the Bioconductor package DNAcopy.
Copy numbers (double deletions, losses, neutral, gains, and
amplifications) that were significantly altered across all samples
were determined using the GISTIC 2.02 algorithm using the
default parameters (33) in the NIH High-Performance Comput-
ing Biowulf cluster computing (http://hpc.nih.gov).
NanoString assay
FFPE tumor cores from the HGSC methylation array set (N ¼
70) were sent to the international ovarian tumor tissue analysis
(OTTA) consortium for gene expression assays of 518 genes on the
NanoString platform analysis. Tissues were deparaffinized with
xylene and processed with the Qiagen miRNeasy FFPE protocol
(Qiagen) as per the manufacturer's recommendation, including
DNase 1 treatment and with an extended proteinase K digest
(55 C digest period extended to 45 minutes). RNA was quanti-
tated on Nanodrop spectrophotometer. Nine samples failed
quality controls, leaving 61 samples for analyses.
Specimens were processed following NanoString gene expres-
sion codeset procedures (NanoString). Briefly, 500 ng of tRNA
from each sample was mixed with a custom codeset (NanoString)
and hybridization buffer (NanoString). Specimens were hybrid-
ized in a Tetrad 2 Thermal Cycler (Bio-Rad) overnight for 16 or
20 hours and then processed on the nCounter Prep Station
(NanoString). Cartridges were then imaged on nCounter Digital
Analyzer (NanoString). Data was normalized to housekeeping
genes (RPL19, ACTB, PGK1, SDHA, and POLR1B) and prepro-
cessed using three pooled ovarian cancer specimens, run on the
same codeset-lot, and using the same protocols, as references in
accordance with a single sample classification scheme outlined
previously (34).
Statistical analysis
Analyses were performed using the R software (version 3.1.1)
and appropriate Biconductor packages.
Consensus nonnegative matrix factorization (NMF) clustering
algorithm (35) was used for unsupervised class discovery using
the 1,000 most variable methylation probes according to the
median absolute deviation (MAD). To identify the number of
stable clusters (rank) in our dataset, we performed 100 indepen-
dent NMF runs for ranks between 2 and 7 using the Lee and Seung
method (36). Previous studies have suggested that 30–50 runs are
sufficient to assess cluster stability (37, 38). For each rank, the
algorithm calculates how frequently two samples are grouped
together in repeated clustering runs. The resulting pairwise con-
sensus rates (cophenetic correlation coefficient) are used for
assessment of cluster stability. To estimate the optimal number
of clusters, we followed the approach proposed by Brunet and
colleagues (37) by selecting the rank for which the cophenetic
correlation coefficient starts to decrease, and therefore the stability
of the cluster starts to decrease. This approach has been previously
used in analyses of ovarian cancer samples (17, 18, 39–41). In
addition, we performed 2,000 bootstrap runs on resampled data
to compute confidence intervals (CI) around each cophenetic
coefficient and to assess whether the optimal rank choice was
statistically significantly different to other ranks. The choice of
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Bodelon et al.
optimal rank was robust to the number of independent NMF runs,
the number of probes, and the NMF algorithm used.
Overall survival was computed as the number of years between
the year of diagnosis and the year of death from all causes, the date
of last follow-up, or 5-year censored survival data. Kaplan–Meier
curves compared overall survival according to subgroups or
histotype and a log-rank test was used to assess the survival
distributions across the subgroups. For univariate and multivar-
iate (adjusted for age, stage, grade, and study) survival analysis, a
Cox proportional hazards regression model was used, and HRs
and 95% CIs were estimated. Year of diagnosis was not associated
with survival.
To quantify the level of genomic instability in each sample, we
computed the fraction of the genome that was altered. This was
defined as the percentage of regions with gains or losses, based on
the GISTIC results, compared with the covered genome. We called
this measure the genomic instability index (GII) of the sam-
ple (42, 43). The Wilcoxon rank-sum test was used to compare
the distribution of the GII values across different groups.
NanoString data for 39 genes were used to classify HGSC
samples according to previously defined gene expression sub-
groups (18) using subtypes scores (44). Briefly, subtype scores
were computed using a weighted score of the genes differentially
expressed in a specific subgroup. Genes upregulated in the sub-
group were allocated a weight of 1, while genes downregulated in
the subgroup were allocated a weight of 1. The subtype score was
computed as the sum of the subtype-specific genes weighted by
the directionality of the gene (44, 45). Because the gene expression
classification approach we used does not take into account batch
variability, we confirmed that the gene expression predicted
clusters were validated against previously identified clusters by
correlating cluster centroids.
Finally, we assessed whether DNA methylation subgroups
added survival information to the previously defined gene expres-
sion subgroups. Using a likelihood ratio test, we compared a
predictor model with gene expression subgroups, age, and stage,
and stratified the baseline hazard by study to a model that had the
same variables and the methylation subgroups.
All statistical tests were two-sided and a P value of less than 0.05
was considered statistically significant.
Results
Our analysis included 162 EOC tumors covering the major
histotypes of EOC (Supplementary Table S1). The majority of
tumors were from the SEER RTR (N ¼ 130, 80%). Principal
component analysis was performed on the methylation beta
values to explore whether methylation clustered according to
factors such as study (POCS vs. SEER RTR), study site (Warsaw,
Lodz, Hawaii, Iowa), year of diagnosis, age at diagnosis, or batch
(Supplementary Fig. S2). None of these factors were related to
methylation differences.
Methylation-based across all EOC tumors
Clustering analysis of the 1,000 most variable methylation
probes across all EOC tumors identified four stable, methyla-
tion-based subtypes based on the decrease of the cophenetic
coefficient (C1, C2, C3, and C4; Fig. 1A and B; Supplementary
Table S3). C1 was predominantly comprised of HGSC tumors
(88%; Fig. 1C; Supplementary Table S4); C2 was predominantly
represented by LMPs (66%) and 27% of invasive serous tumors.
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Almost all LMP serous tumors (97%) were in C2. C3 was pre-
dominantly comprised of clear cell and endometrioid tumors
(74%). Finally, C4 was predominantly comprised of HGSC, high-
grade endometrioid carcinomas, and mucinous tumors. The
concordance between methylation subgroups and histologic sub-
types was approximately 67% (Supplementary Table S5). The
mean age at diagnosis of women was 64.0 years old in C1,
49.6 years old in C2, 55.9 years old in C3, and 59.2 years old
in C4 (C1 vs. C2, P ¼ 1.42  108; C1 vs. C3, P ¼ 0.003; C2 vs. C3,
P ¼ 0.029; C2 vs. C4, P ¼ 0.002; other comparisons were not
statistically significant).
Kaplan–Meier curves of the methylation-based subtypes
showed significantly different overall survival outcomes
for the different subtypes (log-rank test, P ¼ 9.1  107;
Fig. 1D). The survival pattern of each of the methylation-
based subtypes resembled that of the major histologic group
predominantly represented in each methylation-based subtype
(Fig. 1D and E). Compared with C2, the risk of mortality of
women in C1 had a 3-fold higher risk of death (HR ¼ 3.17; 95%
CI, 1.11–9.02; P ¼ 0.031; Table 1). In unadjusted analysis, the
risk of mortality of women in cluster C1 was 6-fold higher
compared with women in C2 (HR ¼ 6.29; 95% CI, 2.78–14.23;
P ¼ 9.99  106; Supplementary Table S6). Similarly, women
in C3 had higher risk of death compared with those in C2 (HR
¼ 3.39; 95% CI, 1.38–8.31; P ¼ 0.008). Women in C3 were also
at higher risk compared with patients in cluster C2, but it was
not statistically significant (Supplementary Table S6). However,
when analyses were adjusted for either histology or other
clinical characteristics (importantly stage and grade), estimates
were greatly reduced and none of the associations remained
significant, suggesting that methylation clusters capture several
of the histologic or clinical phenotypes.
Genomic instability index across all EOC tumors
Copy number was measured in a subset of the EOCs (N ¼ 45;
Supplementary Table S2) comprising serous (N ¼ 15), endome-
trioid (N ¼ 20), clear cell (N ¼ 3), and mucinous (N ¼ 7) tumors
(copy number information was not available for serous LMP
tumors). We computed the fraction of genome that is altered
using the GII for each sample (see Materials and Methods; Fig. 2).
The GII was associated with grade, with lower-grade samples
having lower values of GII and high-grade samples having higher
values of GII [mean GII in low-grade tumors: 0.016 (SD ¼ 0.018),
mean GII in high-grade tumors: 0.050 (0.020); Wilcoxon test P ¼
1.03  105]. Next, we compared the distribution of the GII values
across the methylation-based groups and histologies. The average
GII of methylation cluster C1 was 0.061 (SD ¼ 0.016), of C2 was
0.042 (0.030), of C3 was 0.021 (0.017), and of C4 was 0.044
(0.021). Compared with the GII values in methylation cluster C3,
GII values in the methylation-based subtypes C1 and C4 were
significantly higher (C1 vs. C3: Wilcoxon test P ¼ 7.75  105;
C4 vs. C3: Wilcoxon test P ¼ 0.0037). The relationship between
the GII values and histologies was as expected, with serous tumors
having, on average, higher GII values than the other subtypes:
mean GII of serous tumors: 0.055 (SD ¼ 0.024), mean GII of
endometrioid tumors: 0.025 (0.020); mean GII of mucinous
tumors: 0.040 (0.021); and mean GII of clear cell tumors:
0.051 (0.009). Compared with the GII values of endometrioid
tumors, GII values in serous and clear cell tumors were signifi-
cantly higher (serous vs. endometrioid: Wilcoxon test P ¼ 8.2 
104; clear cell vs. endometrioid: Wilcoxon test P ¼ 0.035).
Clinical Cancer Research
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Methylation and Ovarian Cancer Survival

A B C
Rank = 2 Rank = 3 Rank = 4
1 1 1

1.00
0.8 0.8 0.8
Histology
0.6 0.6 0.6
Clear cell
0.4 0.4 0.4 0.8 Endometrioid
0.2 0.2 0.2 Mucinous

Cophenetic coefficient
0.95
0 0 0
Serous
0.6 Serous LMP

0.4 Grade
High

0.90
Rank = 5 Rank = 6 Rank = 7 Low
1 1 1 0.2
0.8 0.8 0.8 Clusters
0.6 0.6 0.6 C1
C2

0.85
0.4 0.4 0.4
C3
0.2 0.2 0.2
C4
0 0 0

2 3 4 5 6 7
Rank

D E
Kaplan−Meier curves (clusters) Kaplan−Meier curves (histologies)
1.0

1.0
Proportion of women alive

Proportion of women alive

0.8

0.8
0.6

0.6
0.4

0.4

Log−rank test P = 3.28e−06

Clear cell
0.2

0.2

C1 Endometrioid
C2 Mucinous
C3 Log−rank test P = 9.1e−07 Serous
C4 Serous LMP
0.0

0.0

0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time since diagnosis (months) Time since diagnosis (months)

Figure 1.
Unsupervised nonnegative matrix factorization of the 1,000 most variable probes based on the MAD. A, Consensus matrices for rank values from 2 to 7 based on
100 independent NMF runs. B, Cophenetic correlation coefficient for rank values from 2 to 7 with 95% CIs computed by bootstrap. C, Methylation profiles of the
clusters. D, Survival curves for the methylation-derived clusters: C1 (N ¼ 51), C2 (N ¼ 44), C3 (N ¼ 34), and C4 (N ¼ 33). E, Survival for the different histologies:
clear cell (N ¼ 7), endometrioid (N ¼ 33), mucinous (N ¼ 16), serous (N ¼ 76), and serous LMP (N ¼ 30).

Methylation-based across in HGSC tumors
Within HGSC, unsupervised clustering of the 1,000 most
variable methylation probes found three stable methylation-
based subtypes (M1, M2, M3; Fig. 3; Supplementary Table S7).
Overall survival was statistically significantly different between
these methylation subtypes of HGSC, with one subtype having
worse survival than the other two (log-rank test, P ¼ 0.0017).
Compared with patients with tumor samples in the HGSC M1/M3
subtypes, patients in the HGSC M2 subtype had significantly
worse survival after adjusting for age, stage, and study (HR ¼
2.21; 95% CI, 1.12–4.36; P ¼ 0.028; Table 1).
Gene expression subgroups of HGSC
For a subset of HGSC tumors (N ¼ 61), expression data for
39 genes were used to classify previously identified gene expres-
sion subtypes of HGSC (Fig. 4). Survival difference, across the
gene expression subgroups, was consistent with previous reports
(log-rank test, P ¼ 0.023): the mesenchymal (N1) and prolifer-
Table 1. HRs for mortality risk comparison of HGSC methylation clusters
M1/M3 vs. M2 HR (95% CI)
Unadjusted 2.76 (1.48–5.12)
Adjusteda 2.21 (1.12–4.36)
NOTE: Sample size of methylation clusters: M1/M3 (N ¼ 52) and M2 (N ¼ 18).
a
Adjusted for age, stage, and study.
ative (N5) subtypes had the worst survival and the immunore-
active (N2) subtype had the most favorable survival. We studied
the relationship between the 1,000 CpG sites used to define the
methylation-based subgroups and expression of the 39 genes
(Supplementary Fig. S3). None of the 1,000 CpG sites used to
define the methylation signatures were located in the 39 genes.
However, we observed five statistically significant correlations
after adjusting P values by the Bonferroni correction (P values
smaller than 1.28  106; Supplementary Fig. S4). They were all
trans/distal associations. Next, we investigated the relationship
between gene expression signatures and methylation signatures.
The survival patterns observed in the methylation subtypes for all
the HGSC samples were retained in the subset of tumors with gene
expression data (Fig. 4). Approximately, half of the subjects in the
gene expression subtype N5 were in the methylation-based cluster
M2. Most subjects in the NanoString-based clusters N1 and N4
were in the methylation-based cluster M3. Although the N2 group
contained the highest proportion of subjects with the methyla-
tion-based cluster M1, approximately half of those in N2 were in
the methylation-based cluster M3 (Fig. 4; Supplementary Table
P S7). We observed that the NanoString and methylation clusters
0.001 were significantly associated with each other (Fisher exact test, P ¼
0.022 0.0037). Next, we investigated whether the methylation based-
classification could add prognostic information to the gene
expression subtypes. A likelihood ratio test comparing survival

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Bodelon et al.

0.15
A B C1
C2
C3
C4

Chr
1

0.10
Status
Clear cell
1 Figure 2.

GII
Endometrioid
0.5 Mucinous A, Genomic profile of the

0.05
2
Serous segmented data, ordered by GII.
Cluster B, GII distribution by clusters
0 C1 indicated by dots: C1 (N ¼ 8), C2 (N

0.00
3
C2 ¼ 7), C3 (N ¼ 15), and C4 (N ¼ 15);
4
C3 0 10 20 30 40 segments indicate the mean values.
−0.5 C4
5
Subject Wilcoxon rank-test P (C1 vs. C3) ¼
Grade 7.75  105. Wilcoxon rank-test P
6 High
−1 (C3 vs. C4) ¼ 0.0037. Other
Low
7 comparisons were not statistically
8 GII
C significant. C, GII distribution by
0.09 histologies indicated by dots:
9
clear cell (N ¼ 3), endometrioid

0.15
0 Serous Mucinous
10 Endometrioid Clear cell (N ¼ 20), mucinous (N ¼ 7), and
serous (N ¼ 15); segments indicate
11

0.10
the mean values. Wilcoxon rank-
12
test P (serous vs. endometrioid) ¼

GII
13 8.2  104. Wilcoxon rank-test

0.05
14 P (endometrioid vs. clear cell) ¼
15
16 0.035. Other comparisons were not
17
0.00 statistically significant.
18
19
20 0 10 20 30 40
21
22
Subject

models with and without HGSC methylation subgroups (M1/
M3 vs. M2) suggested that adding the methylation subgroups
added prognostic information (P ¼ 0.0072; Table 2).
Discussion
EOC is a heterogeneous disease with different histotypes exhi-
biting distinct clinical and genetic features. Most previous studies
of DNA methylation in EOC have focused on a single histotype or
differences between tumor and putative normal tis-
sues (17, 23, 46). However, the relationship between methylation
patterns and the different pathologic histotypes is not well under-
stood (47). In our analysis, we included the major histologic
subtypes, which led to the identification of four EOC methylation
subgroups with shared molecular phenotypes and a statistically
significant survival difference. These EOC methylation subgroups
may provide an alternative classification of ovarian tumors to
histologic subtypes. The methylation-based subgroups could
represent the origin of the tumors. In particular, evidence seems
to suggest that the majority of HGSCs originate from tubal
intraepithelial carcinoma (TIC; ref. 48), while a subset of HGSCs
may have a truly ovarian origin (48, 49) as is still presumed for
LMP serous tumors (50). We found that one of the methylation
subgroups (EOC methylation subgroup C1) was predominately
represented by HGSCs, potentially being tumors of tubal origin,
while another subgroup (EOC methylation subgroup C2) was
predominantly represented by serous LMP and a small subset of
HGSCs, possibly representing tumors of ovarian origin. Thus,
while the HGSCs in both groups were histologically similar, the
difference in methylation patterns among them could provide
additional insights into etiology or carcinogenesis. Endometrioid
and clear cell ovarian tumors are thought to have a common
origin from orthotopic or ectopic endometrial tissue (6). EOC
methylation subgroup C3 was predominately represented by
endometrioid and clear cell ovarian tumors, which, again, could
reflect their cell of origin. Methylation subgroups could also reflect
other etiologic factors, as endometrioid and clear cell tumors seem
to share many risk factors (11).
We related the EOC methylation subgroups to other molecular
features. In particular, we looked at the level of genomic instability
for each of the methylation-based subgroups. This measure was
related to the grade of the tumor. We found that the fraction of the
altered genome differed across the methylation subgroups with
EOC methylation subgroup C1, which had most of the high-grade
tumors, having a higher average fraction of the altered genome
compared with other methylation subgroups.
Previous studies have suggested that HGSCs are a heteroge-
neous group of tumors at the molecular level (17). We found three
HGSC methylation-based subgroups with differences in survival.
The TCGA also had methylation information to characterize
HGSC. However, it used a lower density methylation array (the
Illumina Infinium Human-Methylation27 BeadChip with
approximately 27,000 CpG sites) and, while the authors found
four methylation groups, they concluded that the clusters dem-
onstrated only modest stability with different clustering yielding
varying methylation subgroups (17). We found robust subgroups
regarding the number of probes, clustering methods, and repeated
clustering runs. The majority of the probes used to define the
methylation subgroups in our study were part of the Methyla-
tion450 BeadChip, but not present on the Methylation27 Bead-
Chip. However, our methylation groups need to be replicated.
In addition to the methylation subgroups, gene expression
subtypes were consistent with previous reports (17, 18, 44). These
gene expression subtypes showed a statistically significant differ-
ence in survival, with previously identified groups that demon-
strated more favorable survival also showing better survival in our
study and vice versa. Previous studies that have multiple sub-
groups based on different genomic characteristics, such as gene

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Methylation and Ovarian Cancer Survival

A Rank = 2 Rank = 3 Rank = 4

B
1 1 1

0.8 0.8 0.8

1.00
0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

Cophenetic coefficient
0.95
0 0 0

0.90
Rank = 5 Rank = 6 Rank = 7
1 1 1

0.8 0.8 0.8

0.85
0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0 0 0
2 3 4 5 6 7
Rank

Figure 3.
Unsupervised nonnegative matrix
factorization based on the 1,000 most C D
variable probes based on the MAD.
A, Consensus matrices for k ¼ 2 to Consensus
k ¼ 7. B, Cophenetic correlation M1
0.8 M2
coefficient for k ¼ 2 to k ¼ 7. M3
C, Methylation profiles of the clusters. 0.6
Kaplan−Meier curves (clusters)

1.0
D, Survival curves for the
0.4

Proportion of women alive

methylation-derived clusters:
M1 (N ¼ 16), M2 (N ¼ 18), and

0.8
0.2
M3 (N ¼ 36).

0.6
0.4
0.2

M1
M2
M3 Log−rank test P = 0.00171
0.0

0 10 20 30 40 50 60
Time since diagnosis (months)

expression and methylation, have not formally looked at the
relationship between the different genomic-based subgroups.
Here, we were interested in understanding what additional sur-
vival information methylation subtypes could add to the gene
expression subtypes. While methylation subgroups were associ-
ated with gene expression subgroups, we observed that methyl-
ation further indicated survival differences beyond current clinical
and molecular factors. This may be important for the classification
of HGSC, a better understanding of their etiology, as well as for
finding markers for early detection, and/or clinical outcomes.
A recent paper found that hypermethylation of six adjacent
CpG loci close to the TAP1 promoter region in 6p21.3 was
associated with shorter time to recurrence in HGSC (51). Five of
the six CpG sites were included in our analyses (cg02181920,
cg06473288, cg24111025, cg25042789, and cg26033526).
Hypermethylation at these five sites was associated with higher
risk of mortality in our study, in agreement with the results of
Wang and colleagues (51). HRs ranged from 3.10 to 5.25 (P values
ranged from 0.11 to 0.062).
Most previous studies of methylation in EOC have used fresh-
frozen ovarian tumors (17, 23). However, the vast majority of
epidemiologic and clinical studies have only archival FFPE tumor
specimens; it is therefore important to investigate whether char-
acterization using methylation is robust in FFPE tumors. During
formalin-fixation, DNA can be damaged or suffer from cross-
linking between biomolecules (DNA to DNA, DNA to proteins,
etc.). The varying age of the tumors and the degree or method of
fixation can also affect the number of artefacts in FFPE sam-
ples (52, 53). While these artifacts can have an impact on several
technologies, such as gene expression or sequencing, DNA meth-
ylation seems to be robust after DNA restoration (54, 55) and
produces accurate, reproducible results. We used tumors from two

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Bodelon et al.

A B Kaplan−Meier curves (clusters)

C
Kaplan−Meier curves (clusters) COL1A2 Methylation
M1

1.0
SPARC 2
1.0

M2
COL3A1
M3

Proportion of women alive

S100A6
Proportion of women alive

FN1 0 Nanostring
N1

0.8
TAGLN
0.8

EZR
N2
N4
SORL1 −2 N5
SLC40A1

0.6
MEST
0.6

ASRGL1 −4
ISG15
CTHRC1

0.4
0.4

DCN
FOLR1
CYP4B1
KIF1A

0.2
0.2

UCHL1
IDO1
N1 M1
N2 M2
PPL
N4 Log−rank test P = 0.000796
Log−rank test P = 0.0227 M3 PDZK1IP1

0.0
N5
0.0

NRIP1
SALL2

0 10 20 30 40 50 60 0 10 20 30 40 50 60 PRTFDC1
CXCL9

Time since diagnosis (months) Time since diagnosis (months) IGHM

IGJ
CXCL10
CXCL11
SLAMF7
ADAMDEC1
CXCL17
SCGB1D2
TSPAN8
C10orf116
MYCN
HMGA2
CD55
ZNF423

Figure 4.
A, Survival curves for gene expression subtypes [N1: mesenchymal (N ¼ 12); N2: immunoreactive (N ¼ 13); N4: differentiated (N ¼ 17); N5: proliferative (N ¼ 19)].
B, Survival curves for methylation clusters among patients with gene expression data: M1 (N ¼ 16), M2 (N ¼ 14), and M3 (N ¼ 31). C, Gene expression heatmap
showing the relationship between methylation subgroups and gene expression subtypes.

distinct studies conducted in two different countries during ovarian cancers (11). Understanding the relationship between
different periods, and we did not observe differences in methyl- risk and genetic factors with molecular subgroups could provide
ation by study. This provides evidence that methylation can additional information that could be crucial for improving pre-
robustly be run in collaborative efforts, such as consortia, where vention, early detection and therapies, especially for HGSC, which
studies are likely conducted in different locations with potentially is the most fatal subtype. We found that HGSC was not a single
variable collection protocols. In our experience, these factors did group at the methylation level. It will be important to understand
not affect the methylation signal. This is important as the classi- the relationship of these HGSC methylation subgroups in relation
fication found in our study could be applied to other epidemi- to risk and genetic factors.
ologic and clinical studies. BRCA status was unknown in our study participants. BRCA
Recent consortial epidemiologic studies have tried to clarify the carriers tend to be diagnosed with HGSC at a younger age than the
etiologic heterogeneity of EOC (11, 56, 57). These studies ana- population average. We explored whether the women diagnosed
lyzed risk factors according to histologic subtypes. Most of the with HGSC before the age of 55 (N ¼ 21) were grouped in specific
known risk factors were more strongly associated with nonserous clusters. We did not observe any specific clustering across those
represented by the different histologic subtypes (C1–C4), but 18
Table 2. HRs and likelihood ratio test comparing survival models of HGSC with of the 21 women were in HGSC cluster M3. In fact, these 18
gene expression clusters and with and without methylation subtypes women represented half of the women in cluster M3. While
N HR (95% CI)a P HR (95% CI)b P promoter methylation of BRCA1 in clusters M1–M3 was not
Gene expression but not methylation subtypes in the model
significantly different, it is possible that these are set of tumors
Gene expression clusters
N1 12 2.38 (0.94–6.05) 0.068 1.60 (0.58–4.45) 0.37
with other BRCA-like properties. Future studies with BRCA status
N2 13 0.75 (0.24–2.28) 0.61 0.81 (0.25–2.59) 0.72 should explore this further.
N4 17 1.00 (ref) — 1.00 (ref) — Our study has several strengths. It is the most comprehensive
N5 19 2.43 (1.02–5.81) 0.045 1.66 (0.62–4.39) 0.31 study of methylation of ovarian cancer to date, as we included the
Gene expression and methylation subtypes in the model major histologic subtypes of ovarian cancer and examined their
Gene expression clusters
shared methylation phenotypes. We used the same laboratory and
N1 12 2.21 (0.87–5.64) 0.097 1.56 (0.57–4.29) 0.39
N2 13 0.72 (0.23–2.20) 0.56 0.71 (0.22–2.31) 0.57
analytical methods for all EOC tumors, allowing for a valid
N4 17 1.00 (ref) — 1.00 (ref) — comparison across subtypes. We also related our methylation-
N5 19 1.44 (0.54–3.86) 0.47 1.13 (0.4–3.13) 0.82 based subgroups to other genomic features, such as copy number
Methylation clusters and gene expression. To date, this has only been done in TCGA,
M1/M3 47 1.00 (ref) - 1.00 (ref) - mostly for HGSC (some low-grade serous carcinomas might have
M2 14 3.05 (1.35–6.88) 0.007 3.12 (1.40–6.98) 0.006
been included as well; ref. 17). However, our study also suffers
Likelihood ratio test 0.0090 0.0072
comparing the
from few limitations, including sample size. Ovarian cancer is a
two models rare disease and most individual studies have small sample sizes.
a
Unadjusted for other variables. We were able to pool data from two distinct studies, and meth-
b
Additional adjusting variables in the model: age, stage and study. ylation showed robust signals independently of the study and
study period. However, larger studies should be conducted in the

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 Published OnlineFirst May 29, 2019; DOI: 10.1158/1078-0432.CCR-18-3720
future to validate our results. While we did not have a replication
set, we evaluated several independent genomic platforms in the
same specimens, including copy number and NanoString with
strong prior data in relation to ovarian cancer. NanoString-based
clustering analysis of HGSC was aligned with previous classifica-
tions of gene expression. Another limitation of our study is the
lack of treatment information. The majority of the tumors includ-
ed in our analyses were from SEER population–based registries for
which specific treatment information is not available. It is possible
that intrinsic characteristics of the tumors could result in better
response to a specific therapy treatment or, independently of
treatment, the molecular characteristics described in our analysis
provide a survival advantage to the patient. Future studies with
treatment information will be able to study this question in detail.
In conclusion, in this comprehensive analysis of ovarian tumor
DNA methylation, we showed that there are four methylation-
defined subgroups of EOC with survival differences, similar to
histologic subtypes. Moreover, within HGSCs, we found meth-
ylation subgroups that added significant survival information to
expression-derived molecular subgroups.
Disclosure of Potential Conflicts of Interest
J.K. Killian is an employee of Foundation Medicine. No potential conflicts of
interest were disclosed by the other authors.
Authors' Contributions
Conception and design: C. Bodelon, J.K. Killian, W.F. Anderson, A. Talhouk,
M. Sherman, N. Wentzensen
Development of methodology: C. Bodelon, J.K. Killian, J.N. Sampson,
M.S. Anglesio, M. Sherman, N. Wentzensen
References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin
2015;65:5–29.
2. K€obel M, Bak J, Bertelsen BI, Carpen O, Grove A, Hansen ES, et al. Ovarian
carcinoma histotype determination is highly reproducible, and is
improved through the use of immunohistochemistry. Histopathology
2014;64:1004–13.
3. Seidman JD, Horkayne-Szakaly I, Haiba M, Boice CR, Kurman RJ, Ronnett
BM. The histologic type and stage distribution of ovarian carcinomas of
surface epithelial origin. Int J Gynecol Pathol 2004;23:41–4.
4. Cho KR, Shih Ie M. Ovarian cancer. Annu Rev Pathol 2009;4:287–313.
5. Koonings PP, Campbell K, Mishell DR Jr, Grimes DA. Relative frequency of
primary ovarian neoplasms: a 10-year review. Obstet Gynecol 1989;74:
921–6.
6. McCluggage WG. Morphological subtypes of ovarian carcinoma: a review
with emphasis on new developments and pathogenesis. Pathology 2011;
43:420–32.
7. Trabert B, Coburn SB, Mariani A, Yang HP, Rosenberg PS, Gierach GL,
et al. Reported incidence and survival of fallopian tube carcinomas: a
population-based analysis from the North American Association of
Central Cancer Registries. J Natl Cancer Inst 2018;110:750–7.
8. Sung PL, Chang YH, Chao KC, Chuang CM, Task Force on Systematic
Review, Meta-analysis of Ovarian Cancer. Global distribution pattern of
histological subtypes of epithelial ovarian cancer: a database analysis and
systematic review. Gynecol Oncol 2014;133:147–54.
9. Gardner GJ, Birrer MJ. Ovarian tumors of low malignant potential: can
molecular biology solve this enigma? J Natl Cancer Inst 2001;93:
1122–3.
10. Zanetta G, Rota S, Chiari S, Bonazzi C, Bratina G, Mangioni C. Behavior of
borderline tumors with particular interest to persistence, recurrence, and
progression to invasive carcinoma: a prospective study. J Clin Oncol 2001;
19:2658–64.
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on July 29, 2019. © 2019 American Association for Cancer
Research.
Methylation and Ovarian Cancer Survival
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): C. Bodelon, W.F. Anderson, R. Matsuno,
J. Lissowska, M.S. Anglesio, D.D.L. Bowtell, J.A. Doherty, S.J. Ramus,
M. Sherman, N. Wentzensen
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): C. Bodelon, J.N. Sampson, L.A. Brinton, J.A. Doherty,
A. Talhouk, M. Sherman, N. Wentzensen
Writing, review, and/or revision of the manuscript: C. Bodelon, J.K. Killian,
W.F. Anderson, R. Matsuno, L.A. Brinton, J. Lissowska, M.S. Anglesio,
D.D.L. Bowtell, J.A. Doherty, S.J. Ramus, A. Talhouk, M. Sherman,
N. Wentzensen
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): C. Bodelon, J. Lissowska, S.J. Ramus,
M. Sherman, N. Wentzensen
Study supervision: C. Bodelon, W.F. Anderson, J. Lissowska
Others (pathology and development of resources): M. Sherman
Acknowledgments
This research was supported by the Intramural Research Program of the NCI.
In addition, S.J. Ramus acknowledges support by the grant "Identifying Prog-
nostic Markers and Therapeutic Targets for Serous Ovarian Cancer" (R01-
CA172404), J.A. Doherty acknowledges support by the grant "Epidemiologic
factors and survival by molecular subtypes of ovarian cancer" (R01-CA168758),
and M.S. Anglesio acknowledges support by the Canadian Institutes of Health
Research (CIHR) Proof of Principle (phase I) grant "PrOTYPE: An Enabling
Technology to Improve Ovarian Cancer Care" and funding from the Janet D.
Cottrelle Foundation Scholar Program administered by the BC Cancer
Foundation.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received November 13, 2018; revised March 18, 2019; accepted May 23,
2019; published first May 29, 2019.
11. Wentzensen N, Poole EM, Trabert B, White E, Arslan AA, Patel AV,
et al. Ovarian cancer risk factors by histologic subtype: an analysis
from the Ovarian Cancer Cohort Consortium. J Clin Oncol 2016;34:
2888–98.
12. Goode EL, Chenevix-Trench G, Song H, Ramus SJ, Notaridou M, Law-
renson K, et al. A genome-wide association study identifies susceptibility
loci for ovarian cancer at 2q31 and 8q24. Nat Genet 2010;42:874–9.
13. Bolton KL, Tyrer J, Song H, Ramus SJ, Notaridou M, Jones C, et al. Common
variants at 19p13 are associated with susceptibility to ovarian cancer.
Nat Genet 2010;42:880–4.
14. Shen H, Fridley BL, Song H, Lawrenson K, Cunningham JM, Ramus SJ, et al.
Epigenetic analysis leads to identification of HNF1B as a subtype-specific
susceptibility gene for ovarian cancer. Nat Commun 2013;4:1628.
15. Raja FA, Chopra N, Ledermann JA. Optimal first-line treatment in ovarian
cancer. Ann Oncol 2012;23:x118–27.
16. Coward JI, Middleton K, Murphy F. New perspectives on targeted therapy
in ovarian cancer. Int J Womens Health 2015;7:189–203.
17. Cancer Genome Atlas Research Network. Integrated genomic analyses of
ovarian carcinoma. Nature 2011;474:609–15.
18. Tothill RW, Tinker AV, George J, Brown R, Fox SB, Lade S, et al. Novel
molecular subtypes of serous and endometrioid ovarian cancer linked to
clinical outcome. Clin Cancer Res 2008;14:5198–208.
19. Kurman RJ, Shih Ie M. Molecular pathogenesis and extraovarian origin of
epithelial ovarian cancer–shifting the paradigm. Hum Pathol 2011;42:
918–31.
20. Kurman RJ, Shih Ie M. The origin and pathogenesis of epithelial ovarian
cancer: a proposed unifying theory. Am J Surg Pathol 2010;34:433–43.
21. Zaino RJ, Brady MF, Lele SM, Michael H, Greer B, Bookman MA. Advanced
stage mucinous adenocarcinoma of the ovary is both rare and highly lethal.
Cancer 2011;117:554–62.
22. Jones PA, Baylin SB. The epigenomics of cancer. Cell 2007;128:683–92.
Clin Cancer Res; 2019 OF9
 Published OnlineFirst May 29, 2019; DOI: 10.1158/1078-0432.CCR-18-3720
Bodelon et al.
23. Cicek MS, Koestler DC, Fridley BL, Kalli KR, Armasu SM, Larson MC, et al.
Epigenome-wide ovarian cancer analysis identifies a methylation profile
differentiating clear-cell histology with epigenetic silencing of the HERG
Kþ channel. Hum Mol Genet 2013;22:3038–47.
24. Garcia-Closas M, Brinton LA, Lissowska J, Richesson D, Sherman ME,
Szeszenia-Dabrowska N, et al. Ovarian cancer risk and common variation
in the sex hormone-binding globulin gene: a population-based case-
control study. BMC Cancer 2007;7:60.
25. Matsuno RK, Sherman ME, Visvanathan K, Goodman MT, Hernandez BY,
Lynch CF, et al. Agreement for tumor grade of ovarian carcinoma: analysis
of archival tissues from the surveillance, epidemiology, and end results
residual tissue repository. Cancer Causes Control 2013;24:749–57.
26. Killian JK, Kim SY, Miettinen M, Smith C, Merino M, Tsokos M, et al.
Succinate dehydrogenase mutation underlies global epigenomic diver-
gence in gastrointestinal stromal tumor. Cancer Discov 2013;3:648–57.
27. Killian JK, Walker RL, Bilke S, Chen Y, Davis S, Cornelison R, et al. Genome-
wide methylation profiling in archival formalin-fixed paraffin-embedded
tissue samples. Methods Mol Biol 2012;823:107–18.
28. Aryee MJ, Jaffe AE, Corrada-Bravo H, Ladd-Acosta C, Feinberg AP, Hansen
KD, et al. Minfi: a flexible and comprehensive Bioconductor package for the
analysis of Infinium DNA methylation microarrays. Bioinformatics 2014;
30:1363–9.
29. Maksimovic J, Gordon L, Oshlack A. SWAN: Subset-quantile Within Array
Normalization for Illumina Infinium HumanMethylation450 BeadChips.
Genome Biol 2012;13:R44.
30. Chen YA, Lemire M, Choufani S, Butcher DT, Grafodatskaya D, Zanke BW,
et al. Discovery of cross-reactive probes and polymorphic CpGs in the
Illumina Infinium HumanMethylation450 microarray. Epigenetics 2013;
8:203–9.
31. Smyth GK, Speed T. Normalization of cDNA microarray data. Methods
2003;31:265–73.
32. Venkatraman ES, Olshen AB. A faster circular binary segmentation algo-
rithm for the analysis of array CGH data. Bioinformatics 2007;23:657–63.
33. Mermel CH, Schumacher SE, Hill B, Meyerson ML, Beroukhim R, Getz G.
GISTIC2.0 facilitates sensitive and confident localization of the targets of
focal somatic copy-number alteration in human cancers. Genome Biol
2011;12:R41.
34. Talhouk A, Kommoss S, Mackenzie R, Cheung M, Leung S, Chiu DS, et al.
Single-patient molecular testing with NanoString nCounter data using a
reference-based strategy for batch effect correction. PLoS One 2016;11:
e0153844.
35. Gaujoux R, Seoighe C. A flexible R package for nonnegative matrix
factorization. BMC Bioinformatics 2010;11:1–9.
36. Lee DD, Seung HS. Algorithms for non-negative matrix factorization. 2001.
https://collaborate.princeton.edu/en/publications/algorithms-for-non-negative-
matrix-factorization
37. Brunet J-P, Tamayo P, Golub TR, Mesirov JP. Metagenes and molecular
pattern discovery using matrix factorization. Proc Natl Acad Sci U S A 2004;
101:4164–9.
38. Hutchins LN, Murphy SM, Singh P, Graber JH. Position-dependent motif
characterization using non-negative matrix factorization. Bioinformatics
2008;24:2684–90.
39. Konecny GE, Wang C, Hamidi H, Winterhoff B, Kalli KR, Dering J, et al.
Prognostic and therapeutic relevance of molecular subtypes in high-grade
serous ovarian cancer. J Natl Cancer Inst 2014;106:pii: dju249.
40. Way GP, Rudd J, Wang C, Hamidi H, Fridley BL, Konecny GE, et al.
Comprehensive cross-population analysis of high-grade serous ovarian
cancer supports no more than three subtypes. G3 2016;6:4097–103.
OF10 Clin Cancer Res; 2019
Downloaded from clincancerres.aacrjournals.org on July 29, 2019. © 2019 American Association for Cancer
Research.
41. Bosquet JG, Marchion DC, Chon H, Lancaster JM, Chanock S. Analysis of
chemotherapeutic response in ovarian cancers using publicly available
high-throughput data. Cancer Res 2014;74:3902–12.
42. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The
genomic and transcriptomic architecture of 2,000 breast tumours reveals
novel subgroups. Nature 2012;486:346–52.
43. Bodelon C, Vinokurova S, Sampson JN, den Boon JA, Walker JL,
Horswill MA, et al. Chromosomal copy number alterations and HPV
integration in cervical precancer and invasive cancer. Carcinogenesis
2016;37:188–96.
44. Leong HS, Galletta L, Etemadmoghadam D, George J, Australian Ovarian
Cancer Society, Kobel M, et al. Efficient molecular subtype classification of
high-grade serous ovarian cancer. J Pathol 2015;236:272–7.
45. Helland Å, Anglesio MS, George J, Cowin PA, Johnstone CN, House CM,
et al. Deregulation of MYCN, LIN28B and LET7 in a molecular subtype
of aggressive high-grade serous ovarian cancers. PLoS One 2011;6:
e18064.
46. Kolbe DL, DeLoia JA, Porter-Gill P, Strange M, Petrykowska HM, Guirguis
A, et al. Differential analysis of ovarian and endometrial cancers identifies a
methylator phenotype. PLoS One 2012;7:e32941.
47. Earp MA, Cunningham JM. DNA methylation changes in epithelial ovarian
cancer histotypes. Genomics 2015;106:311–21.
48. Erickson BK, Conner MG, Landen CN Jr. The role of the fallopian
tube in the origin of ovarian cancer. Am J Obstet Gynecol 2013;209:
409–14.
49. Przybycin CG, Kurman RJ, Ronnett BM, Shih I-M, Vang R. Are all pelvic
(nonuterine) serous carcinomas of tubal origin? Am J Surg Pathol 2010;34:
1407–16.
50. Shih Ie M, Kurman RJ. Molecular pathogenesis of ovarian borderline
tumors: new insights and old challenges. Clin Cancer Res 2005;11:
7273–9.
51. Wang C, Cicek MS, Charbonneau B, Kalli KR, Armasu SM, Larson MC, et al.
Tumor hypomethylation at 6p21.3 associates with longer time to recur-
rence of high-grade serous epithelial ovarian cancer. Cancer Res 2014;74:
3084–91.
52. Kerick M, Isau M, Timmermann B, Sultmann H, Herwig R, Krobitsch S, et al.
Targeted high throughput sequencing in clinical cancer settings: formal-
dehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and
tumor heterogeneity. BMC Med Genomics 2011;4:68.
53. Schweiger MR, Kerick M, Timmermann B, Albrecht MW, Borodina T,
Parkhomchuk D, et al. Genome-wide massively parallel sequencing of
formaldehyde fixed-paraffin embedded (FFPE) tumor tissues for copy-
number- and mutation-analysis. PLoS One 2009;4:e5548.
54. Dumenil TD, Wockner LF, Bettington M, McKeone DM, Klein K, Bowdler
LM, et al. Genome-wide DNA methylation analysis of formalin-fixed
paraffin embedded colorectal cancer tissue. Genes Chromosomes Cancer
2014;53:537–48.
55. de Ruijter TC, de Hoon JPJ, Slaats J, de Vries B, Janssen MJFW, van Wezel T,
et al. Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using
Infinium HumanMethylation450 BeadChip assays. Lab Invest 2015;95:
833–42.
56. Ose J, Poole EM, Schock H, Lehtinen M, Arslan AA, Zeleniuch-Jacquotte
A, et al. Androgens are differentially associated with ovarian cancer
subtypes in the Ovarian Cancer Cohort Consortium. Cancer Res 2017;
77:3951–60.
57. Cuellar-Partida G, Lu Y, Dixon SC, Australian Ovarian Cancer Society,
Fasching PA, Hein A, et al. Assessing the genetic architecture of epithelial
ovarian cancer histological subtypes. Hum Genet 2016;135:741–56.
Clinical Cancer Research
 Published OnlineFirst May 29, 2019; DOI: 10.1158/1078-0432.CCR-18-3720

Molecular Classification of Epithelial Ovarian Cancer Based

on Methylation Profiling: Evidence for Survival
Heterogeneity
Clara Bodelon, J. Keith Killian, Joshua N. Sampson, et al.

Clin Cancer Res Published OnlineFirst May 29, 2019.

Updated version Access the most recent version of this article at:
doi:10.1158/1078-0432.CCR-18-3720

Supplementary Access the most recent supplemental material at:

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