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Tumor and Stem Cell Biology
IDH1 R132H Mutation Generates a Distinct Phospholipid
Metabolite Profile in Glioma
Morteza Esmaeili
1
, Bob C. Hamans
2
, Anna C. Navis
3
, Remco van Horssen
4,5
, Tone F. Bathen
1
,
Ingrid S. Gribbestad
1†
, William P. Leenders
3
, and Arend Heerschap
1,2
Abstract
Many patients with glioma harbor specific mutations in the isocitrate dehydrogenase gene IDH1 that associate
with a relatively better prognosis. IDH1-mutated tumors produce the oncometabolite 2-hydroxyglutarate.
Because IDH1 also regulates several pathways leading to lipid synthesis, we hypothesized that IDH1-mutant
tumors have an altered phospholipid metabolite profile that would impinge on tumor pathobiology. To
investigate this hypothesis, we performed
31
P-MRS imaging in mouse xenograft models of four human gliomas,
one of which harbored the IDH1-R132H mutation.
31
P-MR spectra from the IDH1-mutant tumor displayed a
pattern distinct from that of the three IDH1 wild-type tumors, characterized by decreased levels of phosphoetha-
nolamine and increased levels of glycerophosphocholine. This spectral profile was confirmed by ex vivo analysis of
tumor extracts, and it was also observed in human surgical biopsies of IDH1-mutated tumors by
31
P high-
resolution magic angle spinning spectroscopy. The specificity of this profile for the IDH1-R132H mutation was
established by in vitro
31
P-NMR of extracts of cells overexpressing IDH1 or IDH1-R132H. Overall, our results
provide evidence that the IDH1-R132H mutation alters phospholipid metabolism in gliomas involving phos-
phoethanolamine and glycerophosphocholine. These new noninvasive biomarkers can assist in the identification
of the mutation and in research toward novel treatments that target aberrant metabolism in IDH1-mutant glioma.
Cancer Res; 74(17); 4898–907. 2014 AACR.
Introduction
Diffuse gliomas are the most common malignant brain-born
tumors and are incurable with present therapeutic strategies
(1). These tumors are classified by the World Health Organi-
zation (WHO) as grade 2, 3, and 4 of which grade 4 glioma
(glioblastoma, GBM) is the most malignant type. The current
median survival from the time of diagnosis for GBMs is only
14.6 months and for lower grades between 4 and 15 years (2, 3).
This highly variable survival calls for reliable prognostic bio-
markers for rational decision making in clinical management.
Such biomarkers have become available with the discovery
that in more than 70% of grade 2 and 3 gliomas and in
secondary GBMs, one of the genes for isocitrate dehydrogenase
(IDH1 and IDH2) carry specific mutations, which are associated
with prolonged overall survival (4–8). IDH1, the predominantly
affected enzyme (>95%), catalyzes the conversion of isocitrate
into a-ketoglutarate (a-KG) in the cytosol, using NADP as
electron acceptor to generate NADPH (Fig. 1A). IDH1 can also
catalyze the reductive carboxylation of a-KG to isocitrate that
can be further processed to citrate and acetyl- and succinyl-
CoA, important anabolic precursors for lipid synthesis (9). The
mutation in IDH1, almost always affecting arginine R132,
confers a neomorphic activity to the enzyme, which results
in NADPH-dependent conversion of a-KG to 2-hydroxygluta-
rate (2-HG; Fig. 1A; ref. 10). The mutant enzyme lacks the
capacity of reductive carboxylation (11). As 2-HG accumulates
in mutated tumor cells and tissues (12–14), it has attracted
attention as a potential biomarker in the diagnosis and prog-
nosis of gliomas, in particular as the high levels of 2-HG can
be detected noninvasively by
1
H MR spectroscopy (MRS) in
humans (8, 15–19).
1
H MRS has been explored extensively in the diagnosis and
treatment evaluation of brain tumors in humans (20, 21). MR
spectra of the brain show a single spectral peak for the methyl
protons of small choline compounds, which are involved in the
Kennedy pathway of membrane lipid synthesis and breakdown
(Fig. 1B). In brain tumors, choline metabolism is adapted to the
needs of higher proliferation and to the physiologic microen-
vironment (such as acidic extracellular pH; refs. 22, 23), and
the intensity of this peak (labeled as total choline or tCho) is
often increased (24). Another prominent spectral change is a
1
Department of Circulation and Medical Imaging, Norwegian University of
Science and Technology (NTNU), Trondheim, Norway.
2
Department of
Radiology, Radboud University Medical Center, Nijmegen, the Netherlands.
3
Department of Pathology, Radboud University Medical Center, Nijmegen,
the Netherlands.
4
Department of Cell Biology, Radboud Institute for Molec-
ular Life Sciences, Nijmegen, the Netherlands.
5
Department of Clinical
Chemistry andHematology, St. ElisabethHospital, Tilburg, the Netherlands.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
†Deceased.
W.P. Leenders and A. Heerschap contributed equally to the article.
Corresponding Author: Morteza Esmaeili, Norwegian University of
Science and Technology (NTNU), NTNU/ISB, P.O. Box 8905, 7491,
Trondheim, Norway. Phone: 47-451-22-970 Fax: 47-728-28-372; E-mail:
m.esmaeili@ntnu.no
doi: 10.1158/0008-5472.CAN-14-0008
2014 American Association for Cancer Research.
Cancer
Research
Cancer Res; 74(17) September 1, 2014
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decrease of the peak for the methyl protons of N-acetyl
aspartate (NAA), a neuronal marker compound, reflecting
replacement of neurons by glial tumor cells (25). The tCho/
NAA ratio is, therefore, often used as a biomarker for tumor
load and malignancy in gliomas (26–28). The intensity of the
tCho peak also correlates with cell density, and may be related
to gliosis (29, 30).
To understand in more detail what determines the tCho
peak intensity, an analysis of each contributing component is
needed. This is possible with
1
H MRS of ex vivo biopsy material,
which has a better spectral resolution than in vivo MRS and
allows the separation of tCho into peaks for phosphocholine
(PC), glycerophosphocholine (GPC), and free choline (24). Ex
vivo
1
H MRS or high-resolution magic angle spinning (HR-
MAS) spectroscopy has revealed that PC and GPC contribute
importantly to the increase of tCho in brain tumors and also
uncovered more subtle relationships of choline compounds
with tumor features, in particular with tumor grade (31–34).
Direct in vivo detection of PC and GPC is possible by
31
P
MRS, which also enables detection of phosphoethanolamine
(PE) and glycerophosphoethanolamine (GPE), thereby provid-
ing a more complete picture of in vivo phospholipid metab-
olism (35). Because
31
P MRS is less sensitive than
1
H MRS and
requires dedicated radiofrequency probes, it has been less used
to examine phospholipid metabolites in vivo in brain tumors
(36, 37). However, the increased access to high-field (pre-)
clinical MR scanners, which improves
31
P MRS sensitivity and
resolution, invigorates its further exploration in studies of
tumor phospholipid metabolism.
As a-KG and NADPH are important components for lipo-
genesis (38, 39) and as the mutated IDH enzyme consumes
both compounds and lacks reductive carboxylation capacity,
Figure 1. Schemes of metabolism
involved in 2-HG biosynthesis (A),
and of choline and ethanolamine
phospholipid metabolism (B).
Arrows, metabolic pathways.
A, IDH1/2 catalyze oxidative
decarboxylation of isocitrate to
a-KG using NADP
þ
as a cofactor
to generate NADPH and CO
2
.
Mutations in these genes generate
the oncometabolite 2-HG by
consuming NADPH, and have an
impact on intracellular signaling
and epigenetics. The citrate
generated via the TCA cycle
contributes to the lipid synthesis.
This pathway can be interrupted by
mutations in IDH1 and/or IDH2
genes. Subsequent metabolism of
citrate produces acetyl-CoA for
fatty acid and/or lipid synthesis,
and other intermediates such as
oxaloacetate and malate in TCA
cycle. B, metabolic pathways of
PtdCho and PtdEtn. Acyl-CoA,
acyl-coenzyme A; Etn,
ethanolamine; CDP-Etn,
cytidinediphosphate
ethanolamine; PtdSer,
phosphatidylserine; ChoK, choline
kinase (EC 2.7.1.32); ETNK,
ethanolamine kinase (EC 2.7.1.82);
cPLA2, cytosolic phospholipase
A2 (EC 3.1.1.4); PLC,
phospholipase C (EC 3.1.4.3); PSD,
phosphatidylserine decarboxylase
(EC 4.1.1.65); PSS1,
phosphatidylserine synthase I
(EC Ptdss1); PSS2,
phosphatidylserine synthase II
(EC Ptdss2); PEMT,
phosphatidylethanolamine N-
methyltransferase (EC 2.7.8.29).
Subtyping of IDH1-Mutated Gliomas by
31
P MRS
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we hypothesized that phospholipid metabolism is altered in
IDH1-mutated glioma. To test this hypothesis, we applied in
vivo
31
P MR spectroscopic imaging (MRSI) to four unique and
representative human glioma models growing orthotopically
in mice (40), one carrying the IDH1-R132H mutation (41). The
spectral findings were verified by
31
P NMR analyses of tumor
tissue extracts. To examine the causal relationship of the
spectral profiles to expression of the mutated enzyme, we also
performed
31
P NMR on extracts of glioma cell lines, stably
expressing wild-type or mutated IDH1. Finally, we tested if
similar phospholipid profiles occur in human gliomas by
performing
31
P HR-MAS MRS of biopsies of gliomas in patients
with and without IDH1 mutation.
Materials and Methods
Animals
Balb/c nu/nu mice were obtained from Janvier and housed
in filter-top cages under specific pathogen-free conditions.
Animals were fed a standard diet with food and water ad
libitum. A 12-hour light, 12-hour dark day–night regimen was
applied. All procedures and experiments involving animals
were approved by The National Animal Research Authority,
and carried out according to the European Convention for the
Protection of Vertebrates used for scientific purposes.
Glioma xenografts
Glioma xenografts were injected in the brain of female
Balb/c nu/nu mice (6–10 weeks of age, n>4 for each individual
xenograft line), as described previously (40). Two xenograft
lines, labeled E468 and E473, were originally derived from
human GBM biopsies, whereas E434 and E478 were established
from high-grade oligodendroglioma specimens. The E478
xenograft model contains the heterozygous IDH1-R132H muta-
tion (41). All xenografts grow via diffuse infiltration, whereas
the E434 model additionally presents with some compact
growth (40).
Development of IDH1 (-R132H)–expressing cell lines
To generate IDH1 and IDH1-R132H–expressing U251MG
glioma cell lines, human IDH1 cDNA (accession number,
BC093020, ImaGenes) in pBluescript plasmid was used for
site-directed mutagenesis using the QuickChange Site-Direct-
ed Mutagenesis Kit (Stratagene/Agilent). Primers containing
the critical IDH1-G395A mutation were 50-CCT ATC ATC ATA
GGT CAT CAT GCT TAT GGG GAT CAA TAC AGA GC-30
(forward) and 50-GC TCT GTA TTG ATC CCC ATA AGC ATG
ATG ACC TAT GAT GAT AGG-30(reverse, mutated bases are
underlined). After mutant strand synthesis, DNA of both wild-
type and mutant IDH1 was amplified using primers 50-GAA
TTC ATG TCC AAA AAA ATC AGT GGC GG-30(forward) and
50-GGA TCC TTA AAG TTT GGC CTG AGC-30(reverse) and
cloned into the EcoR1 and BamH1 sites of a customized
pENTR/U6 vector (Invitrogen). Both pENTRY/U6 plasmids
were recombined with pLenti/DEST (Invitrogen) using LR
clonase-II for 6 hours at 25C, proteinase-K treated for 10
minutes at 37C and transformed into One Shot Stbl3 E. coli.
Plasmids from overnight grown colony cultures were isolated
using the Plasmid Midi Kit from Qiagen. To produce lentivirus,
293FT cells were transfected using Lipofectamine 2000 (Invi-
trogen) and the pLenti/DEST DNA was mixed with ViraPower
Packaging mix (Invitrogen). After overnight incubation, medi-
um was replaced and lentivirus-containing medium was har-
vested after 72 hours, filtered, and used to infect U251MG
glioma cells for 6 hours with addition of polybrene (5 mg/mL).
After 48 hours, medium was replaced with medium containing
blasticidin (10 mg/mL; Invitrogen) and cells were kept under
selection for at least 2 weeks. IDH1 expression in U251-IDH1
and U251-IDH1R132H cells was analyzed by Western blotting
using a specific antibody recognizing the mutation (42).
Cells were grown in DMEM containing high glucose, supple-
mented with 10% FCS and penicillin/streptomycin (100 U/mL).
31
P MRS was performed on extracts of U251 cells (parental, IDH-
WT and IDH1-R132H; n¼5 per cell line, >1.5 10
7
cells per
sample) as described below.
Surgical specimens of glioma patients
Surgical specimens were collected from 6 IDH-wild-type
(IDH-WT) glioma patients (4 GBM, 1 anaplastic astrocytoma,
and 1 diffuse astrocytoma), and from 5 IDH1-R132H tumors (3
GBM, 1 anaplastic astrocytoma, and 1 diffuse astrocytoma).
IDH mutation was identified by anti-IDH1-R132H immunos-
taining as described previously (34). Directly after surgical
excision, samples were snap frozen and stored for later analysis
In vivo
31
P 3D MRSI acquisition and analysis
All in vivo MR experiments were performed on a preclinical
7T MR system (Bruker ClinScan) operating at 121.7 MHz for
31
P
MRS. The phosphorus spectra were acquired using a homebuilt
16-mm transmit/receive quadrature coil in combination with a
solenoid
1
H surface coil (20 mm in diameter). The animals were
subjected to MRSI when evident signs of tumor burden (espe-
cially evident weight loss, neurologic defects) were present. A
control group consisting of healthy Balb/c nu/nu animals (n¼
3) was also included. Animals were placed in prone position
and anesthetized by 1.5% isoflurane (Abott) and a mixture of O
2
and N
2
O inhalation. The animal's body temperature was
maintained at 37.5C applying warm air circulation and phys-
iologic monitoring (Small Animal Instrument Inc.) to assess
respiration and temperature. After obtaining a localizer image,
T2-weighted multi spin-echo images in three orthogonal orien-
tations of the brain were acquired. First- and second-order
shimming was performed using FASTMAP (43). The MRSI field
of view (FOV) and matrix size were then selected carefully
reviewing T2-weighted images to cover hyperintense areas
within the tumor tissues (Fig. 2). Three-dimensional
31
PMR
spectroscopy was performed using a 3D MRSI pulse acquire
sequence with an adiabatic BIR-4 45excitation pulse (44), a
repetition time (TR) of 1500 milliseconds, Hanning-weighted
cartesian k-space sampling with 196 signalaverages at the centre
of k-space,2,048 data points overa spectral widthof 4,868 Hz and
a total acquisition time of 2 hours. The FOV of 24 mm 24 mm
24 mm with an 8 88 data matrix and Hamming filtering
resulted in a nominal voxel size of about 5 mm
3
.
After the MR exams, the animals were sacrificed by cervical
dislocation, the brains removed and separated in two halves,
Esmaeili et al.
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which were frozen in liquid nitrogen for subsequent in vitro
MRS analyses. Remaining brain tissue was formalin fixed and
paraffin embedded for further histopathology analysis.
All in vivo MR spectra wereanalyzed using the jMRUI software
(45) and signals fitted with a Lorentzian lineshape, except the
J-coupled signals of ATP, which were fitted with a Gaussian
shape, using the Advanced Method for Accurate, Robust and
Efficient Spectral fitting method (46). Before fitting, spectral
processing was performed, including manual phase correction,
zero-filling (4,096 points), and line-broadening of 20 Hz.
Figure 2.
31
P MRSI of the mouse brain with and without tumor. Orthogonal T2-weighted MR images of a mice brain with an E434 tumor (top, A) and a
normal mouse brain (bottom, B) in axial, coronal, and sagital views, and corresponding
31
PMRspectraof27mm
3
nominal voxels from the 3D
31
P
MRSIdata(CandD).EandF,barplotofthe(PCþGPC þPE þGPE)/ATP (E) and PCr/ATP (F) signal ratios of tumor types growing in mouse
brain (n¼19) and of normal mouse brain (Ctrl, n¼3). Chemical shift is referenced to the GPC resonance at 3.04 ppm. The assigned peaks are (from left
to right); PE, phosphoethanolamine; PC, phosphocholine; Pi, inorganic phosphate; GPE, glycerophosphoethanolamine; GPC, glycerophosphocholine;
PCr, phosphocreatine; ATP, adenosine tri-phosphates. ,P<0.05.
Subtyping of IDH1-Mutated Gliomas by
31
P MRS
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Nuclear magnetic resonance acquisition and analysis of
in vitro and ex vivo samples
Frozen brain tissue samples from the glioma xenografts and
U251 cell pellets were extracted using perchloric acid as describ-
ed in detail previously (47). The neutralized extracts were lyoph-
ilized and kept at 80C until being dissolved in 600 mLofD
2
O.
After final pH adjustments with potassium hydroxide (KOH), in
vitro
31
P NMR spectra of extracts were acquired using a Bruker
spectrometer (Bruker Avance III 600 MHz/54 mm US-Plus)
equipped with a multinuclear QCI CryoProbe (Bruker BioSpin
GmbH) operating at 243.5 MHz for
31
P MRS. High-resolution
31
P
NMR spectra of the water-soluble metabolites were obtained
with proton decoupling during acquisition, a 30flip angle, 8,192
free induction decays (FID), TR ¼4 seconds, spectral width of
14,577 Hz into 36,864 data points in time domain.
31
P HR-MAS spectroscopy was carried out using a 600-MHz
spectrometer (Bruker Avance III 600 MHz/54 mm US-Plus)
equipped with a triple
1
H/
13
C/
31
P MAS probe (Bruker BioSpin
GmbH). The frozen specimens from human brain tumors were
thawed and cut on an ice-pad. Tissue samples were gently
loaded into 30-mL disposable inserts filled with 3 mL
2
H
2
O
(Sigma-Aldrich GmbH) for the
2
H lock. The inserts were then
placed into a 4-mm diameter ZrO
2
MAS rotor (Bruker BioSpin
GmbH). The MAS rotors were spun at 5 kHz and maintained at
4C to minimize enzymatic activities within the tissue samples.
All in vitro and ex vivo spectra were processed using the
Bruker TopSpin V3.0 software (Bruker BioSpin GmbH). The
accumulated FIDs were Fourier transformed after application
of 3 Hz exponential line broadening. Automatic phase and
linear baseline corrections were performed. The GPC peak (at
3.04 ppm) in
31
P MR spectra was used as references for
chemical shift calibration. Following standard processing,
peak areas of phosphorylated metabolites were calculated by
peak fitting (PeakFit V4.12; SeaSolve Software Inc.) using a
combination of Gaussian–Lorentzian lineshapes (Voigt area).
Metabolite concentrations were calculated from peak areas.
Statistical analysis
The difference in the mean value of selected metabolite
ratios were statistically assessed using a two-tailed unpaired
Mann–Whitney test (Prism GraphPad V 4.03 Software Inc.)
and the differences were considered statistically significant
for Pvalues <0.05. All results are represented as mean
standard deviation (SD).
Results
In vivo 3D
31
P MRSI of human glioma xenografts
Tumors in the brain present as hyperintense signal areas on
T2-weighted MR images (compare the tumor-containing brain
300
200
100
0
–100
300
200
100
0
–100
300
200
100
0
–100
300
200
100
0
–100
10 5 0 5 –10 –15 8 7 6 5 4 3
876543
876543
876543
10 5 0 5 –10 –15
10 5 0 5 –10 –15
10 5 0 5
Chemical shift (ppm) Chemical shift (ppm)
–10 –15
20
× 105
× 105
× 106
× 105
10
0
8
6
4
2
0
4
2
0
–2
8
6
4
2
0
–2
PC
AB
GPE
Pi
ATP
PC
PC
PCr
GPC E478
PE
GPC
GPCPE
PE
PE
PE
PC
GPE
GPC
GPE
PE
PC GPC
GPC
GPE
PC
E468
E434
E473
PE
PE
GPC
Pi
Pi
Pi
Pi
GPE
GPE
GPC
GPE
PC
PC
GPE
Figure 3. IDH1-mutated E478
xenografts show a distinct
31
P-
spectral pattern. A, in vivo
31
PMR
spectra obtained from four human
glioma xenograft tumor lines
growing in the mouse brain
(IDH1-mutated xenograft E478,
and IDH1-WT E434, E473, and
E468). In the IDH1-mutated
xenograft, GPC is highly elevated
and PE decreased compared with
the wild-types. B, representative in
vitro 243.5 MHz (
1
H-decoupled)
31
P
MR spectra of tissue extracts of
these tumors; from top to bottom
the IDH1-mutated E478, and the
IDH1-WT lines E434, E468, and
E473. The chemical shift reference
is the GPC resonance at 3.04 ppm.
Esmaeili et al.
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in Fig. 2A with the normal brain in Fig. 2B), and we used these
images for voxel positioning. The
31
P MR spectra of voxels of
interest selected from the 3D MRSI dataset of this brain
showed resolved resonances for a number of compounds,
including ATP, phosphocreatine (PCr), GPC and GPE, inor-
ganic phosphate (Pi), PC, and PE. The
31
P-spectral profiles of
tumor voxels in all four xenograft models differed from those
obtained from voxels in comparable brain areas in non–tumor-
bearing animals (compare Fig. 2C with 2D). The relative
phosphor signal integrals of choline and ethanolamine com-
pounds were increased, as represented by a significantly higher
(PC þGPC þPE þGPE)/ATP ratio for all tumor types (P<
0.05; Fig. 2E). This total relative phospholipid content was not
different among the tumor models. In addition, a significantly
decreased PCr/ATP signal ratio was observed in tumor tissues
compared with the healthy mouse brain tissues (P<0.05;
Fig. 2F).
Among the four human glioma lines, the E478 tumor exhib-
ited a deviating spectral profile in the 2 to 8 ppm range (Fig. 3A
and B). For a quantitative assessment, we first determined for
each metabolite resonance its integral normalized to the sum
of those of all phospholipid metabolite resonances (see Fig. 4A
and B). This revealed a significant decrease of the PE resonance
of the IDH1-mutant E478 xenograft compared with those of the
IDH1-WT xenografts (P¼0.003). A significant increase was
observed for the GPC resonance of E478 compared with IDH1-
WT tumors (P¼0.003). Furthermore, the PC peak of E478
showed a trend for an increase compared with the PC of the
other tumors (P¼0.08). The GPE resonance did not differ
between the tumors. In concordance with the in vivo results, we
observed very similar differences between
31
P NMR spectra of
tumor extracts from IDH1-mutant and wild-type xenografts
(Fig. 3B). Again, PE was reduced and GPC increased in E478
extracts compared with those of the other models (P¼0.004
and 0.01 respectively; Fig. 4B)
These findings suggest that sensitive biomarkers associ-
ated with the presence of IDH1 mutations would be repre-
sented by the peak ratios PC/PE, GPC/GPE, GPC/PE, and
(PC þGPC)/(PE þGPE). A quantitative assessment of mean
in vivo metabolite ratios obtained from all xenograft models
showed that these ratios in the E478 xenograft were more
than 2-fold higher than those of the other xenografts (P<
0.01 for all ratios; Fig. 4C). Similar findings were observed for
these ratios obtained from spectra of tumor tissue extracts
(Fig. 4D).
Phospholipid metabolite ratios involving PE and GPC
are similarly altered in cell lines and human glioma with
an IDH1 mutation
To investigate how specific the phospholipid metabolite
changes are for the IDH1 mutation, we measured in vitro
31
P NMR spectra of extracts of a set of U251-MG cell lines
overexpressing either wild-type or mutant IDH1 (see Fig. 5A–
C). The IDH1-R132H–expressing U251-MG cells showed
Figure 4. Relative tissue levels of
MR detectable phospholipids in
brain xenograft models. Metabolite
signal integrals normalized to the
total integral of detectable
phospholipids signals in
31
PMR
spectra obtained from (A) in vivo
mutant glioma xenograft E478
(n¼4) and wt xenografts (n¼5for
each) and (B) from tissue extracts of
xenograft tissue extracts (n¼4for
E478 and n¼5 for the other tumor
lines). Values are presented as
mean SD. Scatter plot of
metabolite ratios obtained from
31
P spectra measured (C) from
tumors in mouse brain and (D) from
extracted tumor tissue. The
metabolite ratios of IDH1-WT tumor
lines; E473 þE468 þE434 (orange,
n¼15) were compared with the
IDH1-mutated tumor line E478
(green, n¼4). The y-axis values
indicate the mean and SD;
,P<0.01.
Subtyping of IDH1-Mutated Gliomas by
31
P MRS
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significantly higher PC/PE and GPC/GPE levels than U251-
IDHwt (P<0.001) and U251-MG control cell lines (P<0.01 for
both ratios; Fig. 5B). The U251-R132H also showed a signifi-
cantly higher (PC þGPC)/(PE þGPE) ratio (P<0.01; Fig. 5B).
Finally, we investigated if these IDH-mutant specific spectral
findings could be validated in human gliomas. For this purpose,
we obtained
31
P HR-MAS spectra of human glioma specimens.
Again,
31
P spectral profiles in IDH1-mutant gliomas (Fig. 5D–F)
were obtained that were characterized by significant eleva-
tions in PC/PE, GPC/GPE, and (PC þGPC)/(PE þGPE) ratios
(P<0.01 for all ratios) as compared with IDH1-WT glioma
specimens (Fig. 5E).
Discussion
In this study, we tested the hypothesis that IDH1 mutations
affect the phospholipid metabolite profile of tumors by using
IDH1-R132H E478 xenografts (41). Our major finding is that
gliomas with an IDH1 mutation indeed have a phospholipid
profile, which differs from that in gliomas with wild-type IDH1.
This is reflected most clearly in relatively higher GPC and lower
PE tissue levels, resulting in more than two-fold higher PC/PE,
GPC/GPE, and GPC/PE ratios for the IDH1 mutants.
31
P
nuclear magnetic resonance (NMR) of tumor tissue extracts
obtained from the same animals confirmed the in vivo findings.
Moreover,
31
P NMR of extracts of U251 cells, stably expressing
recombinant IDH1-R132H also yielded increased PC/PE and
GPC/GPE ratios compared with IDH-WT cells, proving
that these changes are related to the IDH1 mutation. Finally,
31
P HR-MAS of surgical biopsies of human brain tumors
identified similar IDH mutation–specific phospholipid metab-
olite patterns.
An increased GPC level has been detected by mass spec-
trometry in oligodendroglioma cells expressing the IDH1
mutation as compared with wild-type cells (14), and a positive
correlation of GPC with 2HG was found by HR-MAS of ex vivo
Figure 5.
31
P NMR spectra of U251MG cell extracts and
31
P HR-MAS MR spectra of surgical biopsies from patients with glioma. A, representative
31
PNMR
spectra of cell extracts show similar
31
P-spectral features as observed for the in vivo growing brain xenografts. Relatively decrea sed PE and increased GPC
resonances identify the mutant cell line (U251-R132H) from wild-type cell lines (U251-WT) and U251MG control cells (U251). The green and orange colored
spectra are shifted to the right for a better visualization. B, the PC/PE, GPC/GPE, and (PC þGPC)/(PE þGPE) ratio s are significantly increased in the U251-
R132H cell line compared with the U251-WT and U251 cells. C, phospholipid levels measured from
31
P MR spectra of U251 cell extracts. D, representative
31
P HR-MAS MR spectra of glioma patient biopsies. E, the levels of PC/PE, GPC/GPE, (PC þGPC)/(PE þGPE) ratios in IDH1-R132H glioma patients
are consistent with preclinical results. F, phospholipid metabolite levels measured in glioma patient tissues samples. The y-axis values indicate the mean and
SD; ,P<0.05; ,P<0.01.
Esmaeili et al.
Cancer Res; 74(17) September 1, 2014 Cancer Research
4904
on October 29, 2015. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
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biopsies of low-grade tumors with the mutation (18). Elevated
GPC has been associated with grade 2 and 3 gliomas (32–34).
Because the majority of these have the IDH1 mutation and
elevated GPC is associated with 2-HG (18), this suggests that
GPC levels have a causal relationship with the mutation, which
is in agreement with our findings. There is much less known for
PE. Its resonances in
1
H MR spectra are not well resolved. Data
from
31
P HR-MAS studies of biopsies indicate that PE is
decreased in low-grade gliomas (34, 48), but the only report
describing PE in relation to the IDH1 mutation indicates a
positive correlation with 2-HG, as detected by
1
H HR-MAS (18).
As
31
P NMR of the GBM cell line GS-2 also showed low PE to
PC and high GPC to GPE ratios (49), it would be of interest to
investigate whether this line also carries the IDH mutation.
For all tumors in the mouse brain, we detected a decreased
PCr/ATP ratio, which is in line with findings in
31
P MRS of
human tumors and indicates an altered energy metabolism
(37, 50).
IDH1 mutations are confined to low-grade and secondary
high-grade glioma GBM (4, 5) and confer a relatively good
prognosis to patients suffering from these tumors (6, 7, 51).
Despite their frequent occurrence, glioma xenografts carrying
these mutations are very scarce (41, 52, 53), and in vitro
propagation of IDH1-mutated glioma cell lines is challenging
(54). Interestingly, and in line with clinical observations, E478
xenografts present with lower proliferation rates than IDHwt
counterparts, as established via the Ki67 index, and mice
carrying these xenografts have a longer survival time than
mice carrying IDHwt xenografts (see Supplementary Materi-
als). A correlation between longer survival, lower Ki67, and
lower PC/GPC ratio has also been observed in human patients
with glioma (32, 34).
31
P MRSI of brain tumors and imaging biomarkers for
IDH1 mutation
In this study, we demonstrate that
31
P MR spectra of the
mouse brain can be obtained with a good signal-to-noise ratio
(SNR) by 3D MRSI on a 7T magnet with a dedicated
31
P coil.
Moreover, at this field strength, resolved signals for individual
choline- and ethanolamine-containing metabolites can be
detected. This is also possible for human brain at clinical field
strengths (1.5–3T) using
1
H decoupling (35, 37) and
1
H-
31
P
polarization transfer to enhance sensitivity (55, 56). Thus, it is
worthwhile to investigate if ratios of PE and GPC signals can be
used as biomarkers to assist in the noninvasive metabolic
characterization of IDH mutations in patients with glioma
brain tumors. This may be used in the assessment of the effect
of inhibitors of the mutated IDH enzyme (57).
It is known that human glioma cells with an IDH1 mutation
accumulate 2-HG (12–14). We confirmed the presence of 2-HG
in our E478 model and in IDH-R132H–overexpressing U251
cells, by both LC/MS (41) and 1D and TOCSY
1
H MRS (see
Supplementary Materials). This accumulation enables the
detection of 2-HG by
1
H MRSI, by which patients with this
mutation can be identified (15, 17). However, spectral overlap
hampers discrimination of 2-HG signals from those of gluta-
mine, glutamate, and gamma-amino butyric acid, even with
peak fitting using prior knowledge and optimal echo times.
This may be overcome by spectral editing or 2D
1
H MRS, but at
the expense of increased complexity and longer scan times or
low SNR (17). Moreover, in clinical practice, all these methods
to detect 2-HG may fail due to suboptimal field homogeneity.
As the resolved detection of
31
P signals is less prone to field
inhomogeneities, the distinct phospholipid metabolite profiles
in IDH1-mutated gliomas may have a role in the identification
of this mutation.
Biologic meaning of the change in phospholipid levels
in IDH1-mutated glioma
Many enzymes that are involved in lipid biosynthesis depend
on appropriate levels of cytosolic NADPH and acetyl-CoA. The
activity of IDH1 is an important source for cytosolic NADPH in
the brain (ref. 51; see also Fig. 1A) and only for this reason,
mutations in this enzyme are expected to affect lipid synthesis.
This impact will be augmented by the fact that IDH1 is also
involved in the reductive carboxylation of a-KG to isocitrate,
especially under hypoxic conditions, isocitrate being the build-
ing block for lipids via generation of acetyl- and succinyl-CoA. In
mutated IDH1, this activity is lost (11). Evidently, according to
this model, IDH1-mutated tumor cells are subject to high
metabolic stress, and cells need to adapt to this stress to
facilitate survival and tumor progression (58). In a previous
report we described that E478 xenografts, despite the IDH1
mutation, do not have significantly decreased a-KG levels and
present with densely packed mitochondria. On the basis of these
findings, we postulated that IDH1-mutated tumor cells rescue
the IDH1 defect by upregulating mitochondrial biosynthesis and
concomitantly IDH2 activity, followed by transportof mitochon-
drial a-KG and NADPH to the cytosol (41). To accommodate
mitochondrial biosynthesis, membrane synthesis is required.
The alteredsteady-state levelsof some phospholipid metabolites
in IDH1-mutated gliomasmay be related torapid incorporation
of precursors in phosphatidylcholine (PtdCho) and phosphati-
dylethanolamine (PtdEtn), the most abundant membrane com-
pounds (59). An interesting question that directly follows, is
whether energy production in IDH1-mutated gliomas is bal-
anced more toward oxidative phosphorylation than to aerobic
glycolysis (the Warburg phenomenon; ref. 60). Such considera-
tions may eventually lead to therapeutic handles.
In conclusion, we provide evidence that IDH1 mutations
result in distinct alterations in lipid metabolism that can be
detected noninvasively by
31
P MRSI. These may serve as a
complementary biomarker to characterize the metabolic sta-
tus of IDH1-mutated gliomas during evaluation of anticancer
targeted therapies and in tumor diagnosis. Increased avail-
ability to higher field strength MR systems (3 and 7 T) and
dedicated
31
P coils hold promise for clinical translation of the
31
P MRSI method. Further research is needed to fully elucidate
the roles of PE and GPC, such as their involvement in mito-
chondrial membrane synthesis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: M. Esmaeili, W.P. Leenders, A. Heerschap
Development of methodology: M. Esmaeili, W.P. Leenders, A. Heerschap
Subtyping of IDH1-Mutated Gliomas by
31
P MRS
www.aacrjournals.org Cancer Res; 74(17) September 1, 2014 4905
on October 29, 2015. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst July 8, 2014; DOI: 10.1158/0008-5472.CAN-14-0008
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M. Esmaeili, B.C. Hamans, A.C. Navis, R. van Horssen
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): M. Esmaeili, B.C. Hamans, A.C. Navis, T.F. Bathen,
W.P. Leenders, A. Heerschap
Writing, review, and/or revision of the manuscript: M. Esmaeili, B.C.
Hamans, A.C. Navis, T.F. Bathen, I.S. Gribbestad, W.P. Leenders, A. Heerschap
Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): M. Esmaeili, I.S. Gribbestad,
A. Heerschap
Study supervision: M. Esmaeili, T.F. Bathen, I.S. Gribbestad, W.P. Leenders,
A. Heerschap
Acknowledgments
The authors thank A. Veltien and J. van der Laak for technical assistance
with MR measurements and KS400 software, respectively, and Marieke
Willemse for generating the U251-cell lines. The authors also thank Jeroen
Mooren and Bianca Lemmers van de Weem for their assistance during the
animal experiments.
Grant Support
This research was also supported by NWO investment grants 91106021 and
BIG (VISTA).
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 January 6, 2014; revised April 28, 2014; accepted May 26, 2014;
published OnlineFirst July 8, 2014.
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P MRS
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Morteza Esmaeili, Bob C. Hamans, Anna C. Navis, et al.
Profile in Glioma
IDH1 R132H Mutation Generates a Distinct Phospholipid Metabolite
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