JNCI | Articles 143
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Malignant glioma is the most common form of primary malignant
brain tumor and the glioma histological subtypes include glioblas-
tomas, grades 2 and 3 astrocytomas, grades 2 and 3 oligodendro-
gliomas, grades 2 and 3 oligoastrocytomas, ependymomas, and
pilocytic astrocytomas (1). Presently, there are limited treatment
options for glioma; glioblastoma, the most common glioma
subtype, remains an incurable disease with a median survival of
15 months, even with radiation and temozolomide therapy (2).
A comprehensive appreciation of the integrated genomics and
epigenomics of glioma is needed to better understand the multiple
cellular pathways involved in their development, establish markers
of resistance to traditional therapies, and contribute to the devel-
opment of targeted therapies. Epigenetic alterations can alter gene
expression, gene expression potential, or the regulation of gene
function, and thereby contribute to gliomagenesis. Arguably, the
most widely studied epigenetic mark is DNA methylation that
occurs at cytosine residues in the context of CpG dinucleotides.
Approximately half of human genes have concentrations of CpGs
in their promoter regions and the methylation state of these and
other gene-associated CpGs are widely regarded as critical indica-
tors of gene regulation.
Since 2008, sequencing of gliomas has identified mutations in
the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes
(3–5). The IDH1 and IDH2 enzymes convert isocitrate to alpha
(a)-ketoglutarate producing NADPH and participate in cellular
metabolic processes such as glucose sensing, lipid metabolism, and
oxidative respiration [reviewed in (6)]. Mutations in IDH1 are
consistently found in codon 132 for arginine (R132), and mutations
DNA Methylation, Isocitrate Dehydrogenase Mutation, and
Survival in Glioma
Brock C. Christensen, Ashley A. Smith, Shichun Zheng, Devin C. Koestler, E. Andres Houseman, Carmen J. Marsit,
Joseph L. Wiemels, Heather H. Nelson, Margaret R. Karagas, Margaret R. Wrensch, Karl T. Kelsey, John K. Wiencke
Manuscript received April 14, 2010; revised November 5, 2010; accepted November 8, 2010.
Correspondence to: John K. Wiencke, PhD, Department of Neurological Surgery, Helen Diller Family Cancer Center, University of California San Francisco,
San Francisco, CA 91458 (e-mail: firstname.lastname@example.org).
Background Although much is known about molecular and chromosomal characteristics that distinguish glioma histological
subtypes, DNA methylation patterns of gliomas and their association with other tumor features such as muta-
tion of isocitrate dehydrogenase (IDH) genes have only recently begun to be investigated.
Methods DNA methylation of glioblastomas, astrocytomas, oligodendrogliomas, oligoastrocytomas, ependymomas, and
pilocytic astrocytomas (n = 131) from the Brain Tumor Research Center at the University of California San
Francisco, as well as nontumor brain tissues (n = 7), was assessed with the Illumina GoldenGate methylation
array. Methylation data were subjected to recursively partitioned mixture modeling (RPMM) to derive methyla-
tion classes. Differential DNA methylation between tumor and nontumor was also assessed. The association
between methylation class and IDH mutation (IDH1 and IDH2) was tested using univariate and multivariable
analysis for tumors (n = 95) with available substrate for sequencing. Survival of glioma patients carrying mutant
IDH (n = 57) was compared with patients carrying wild-type IDH (n = 38) using a multivariable Cox proportional
hazards model and Kaplan–Meier analysis. All statistical tests were two-sided.
Results We observed a statistically significant association between RPMM methylation class and glioma histological
subtype (P < 2.2 × 10216). Compared with nontumor brain tissues, across glioma tumor histological subtypes, the
differential methylation ratios of CpG loci were statistically significantly different (permutation P < .0001).
Methylation class was strongly associated with IDH mutation in gliomas (P = 3.0 × 10216). Compared with glioma
patients whose tumors harbored wild-type IDH, patients whose tumors harbored mutant IDH showed statisti-
cally significantly improved survival (hazard ratio of death = 0.27, 95% confidence interval = 0.10 to 0.72).
Conclusion The homogeneity of methylation classes for gliomas with IDH mutation, despite their histological diversity,
suggests that IDH mutation is associated with a distinct DNA methylation phenotype and an altered metabolic
profile in glioma.
J Natl Cancer Inst 2011;103:143–153
144 Articles | JNCI Vol. 103, Issue 2 | January 19, 2011
in IDH2 consistently occur at the analogous amino acid R172 (3,7).
Mutations in IDH1 and IDH2 (IDH when referring to both) are
unlike most cancer-associated enzyme mutations because they
confer neomorphic enzyme activity rather than inactivating, or
constitutively activating, the enzyme. The mutant form of IDH
enzymes convert a-ketoglutarate to 2-hydroxyglutarate in an
NADPH-dependent manner, and via an unknown mechanism
contribute to the pathophysiology of gliomas and leukemias
(5,7,8). IDH mutations occur in approximately 80% of grades 2–3
gliomas and secondary glioblastomas, but less than 10% of primary
glioblastomas (4,5). In gliomas, IDH mutation has been associated
with genetic alterations in other genes including tumor suppres-
sors and oncogenes (5). IDH mutation also has been associated
with younger age and improved survival in glioma patients (5,9).
The somatic genetic signature of any individual tumor is critical
to assessing its clinical and etiologic character. Similarly, the pro-
file of somatic epigenetic alterations is central to forming a com-
plete understanding of the pattern of disrupted cellular functioning
responsible for the deadly behavior of gliomas. Major advances in
the clinical role of epigenetics in gliomas include the findings that
promoter methylation silencing of the O-6-methylguanine-DNA
methyltransferase (MGMT) gene is associated with response to
temozolomide treatment (10). Epigenetic silencing of MGMT
gene is found in approximately 80% of gliomas with mutant IDH1,
compared with approximately 60% of gliomas with wild-type
IDH1 (9). Other common alterations in gliomas are mutations in
tumor protein p53 (TP53) (11) and amplification of the epidermal
growth factor receptor (EGFR) oncogene (12). Better definitions
of the somatic nature of gliomas should integrate both their ge-
netic and epigenetic alterations. In this study, we assessed CpG
methylation patterns, IDH mutation, TP53 mutation, and EGFR
amplification in histologically diverse gliomas to define epigenetic
subgroups of potential clinical and etiologic relevance.
Patients, Materials, and Methods
Patients and Tissue Samples
Fresh frozen tumor tissues of patients (n = 131) diagnosed with
glioma between 1990 and 2003 were obtained from the University
of California San Francisco (UCSF) Brain Tumor Research Center
Tissue Bank. Tumors were previously reviewed by UCSF neuro-
pathologists to assign histological subtypes and grades according to
the World Health Organization classification for patients operated
on at the UCSF Medical Center (1). Tumor samples were defined
as secondary glioblastoma if the patients had previous histological
diagnosis of a lower-grade glioma. Nontumor brain tissue samples
were obtained from cancer-free patients (n = 7) who underwent
temporal lobe resection for treatment of epilepsy at the UCSF
Medical Center. Patient ages were documented at the time of ini-
tial diagnosis. Other demographic and survival data were obtained
from UCSF patient records and the California Cancer Registry.
The Institutional Review Board approval certification was obtained
from the UCSF Committee on Human Research, and subjects
provided written informed consent for tissue collection.
Cell Lines, Cell Culture, and Reagents
A431 cells (a human epidermoid cancer cell line that is known to
have EGFR amplification and overexpression) and HT29 cells
(a human colon adenocarcinoma cell line without EGFR amplifica-
tion) were obtained from American Type Culture Collection
(Manassas, VA). Cell lines were maintained in Dulbecco’s modi-
fied Eagle medium and RPMI 1640 medium (both from Invitrogen,
Carlsbad, CA), respectively, with 10% fetal bovine serum (Hyclone,
Logan, UT) at 37°C in 5% CO2. When cultures reached 80%
confluency, cells were harvested for DNA extraction.
DNA Extraction, Bisulfite Modification, and Methylation
Genomic DNA from 131 glioma tissue samples and seven
nontumor brain tissue samples was isolated from approximately
25 mg wet weight of each frozen tissue sample using QIAamp DNA
mini kit (Qiagen, Inc, Valencia, CA) according to the manufactur-
er’s instructions. DNA was eluted twice in a total of 100 µL of elu-
tion buffer. The same DNA extraction method was applied to A431
and HT29 cell lines that served as EGFR amplification controls.
For DNA methylation analysis, 1 µg of genomic DNA was first
subjected to bisulfite modification using the EZ DNA Methylation
Kit (Zymo Research Corporation, Orange, CA) according to the
manufacturer’s instructions. Bisulfite modification converts
unmethylated cytosine residues to uracil and preserves methylated
cytosine residues as cytosines.
CONTEXT AND CAVEATS
Human gliomas often have mutations in the isocitrate dehydroge-
nase genes (IDH1 and IDH2). IDH mutation is associated with
improved survival in glioma patients. Epigenetic alterations like
DNA methylation at CpG dinucleotides play an important role in
gene regulation. Integration of genetic and epigenetic data is
important for a better understanding of glioma development.
DNA methylation profile of CpG loci and methylation class of 131
glioma and seven non-glioma brain tissues were determined. The
association between IDH mutation and methylation class was ana-
lyzed. Survival analysis of patients carrying IDH mutation vs wild-
type IDH was also performed.
CpG loci showed differential methylation between glioma and non-
glioma tissues. Statistically significant associations were found
between DNA methylation class and histological subtypes and
between DNA methylation class and IDH mutation of gliomas.
Patients carrying IDH mutation in gliomas showed improved sur-
vival compared with patients carrying IDH wild-type after adjust-
ment for age and grade-specific tumor histology.
A distinct methylation pattern in glioma tissues is associated with
Mutation data were not available for all tissue samples, which may
have limited the statistical power of the analyses.
From the Editors
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National Institute of Health (R01CA52689 to M.R.W.; P50CA097257
to M.R.W. and J.K.W; R01CA078609, R01CA121147, R01CA126939,
and R01CA100679 to K.T.K.; R01ES06717 and R01CA126831 to J.K.W.;
P30CA077598 to H.H.N.); Tobacco-Related Diseases Research Program
(18CA-0127 to J.L.W.).
B. C. Christensen and A. A. Smith contributed equally to the work.
M. R. Wrensch, K. T. Kelsey, and J. K. Wiencke are joint lead investigators.
The funders did not have any role in the study design, collection of data,
interpretation of the results, preparation of the article, or the decision to submit
the article for publication.
Affiliations of authors: Department of Pathology and Laboratory Medicine
(BCC, AAS, CJM, KTK) and Department of Community Health (BCC, DCK,
EAH, KTK), Brown University, Providence, RI; Department of Neurological
Surgery, Helen Diller Family Cancer Center (SZ, MRW, JKW) and Department
of Epidemiology and Biostatistics (JLW), University of California San Francisco,
San Francisco, CA; Department of Biostatistics, Harvard School of Public
Health, Boston, MA (EAH); Masonic Cancer Center, Division of Epidemiology
and Community Health, University of Minnesota, Minneapolis, MN (HHN);
Section of Biostatistics and Epidemiology, Department of Community and
Family Medicine, Dartmouth Medical School, Lebanon, NH (MRK).