Brain Tumor Pathol
DOI 10.1007/s10014-011-0023-7

ORIGINAL ARTICLE

Detection of IDH1 mutation in human gliomas: comparisonof immunohistochemistry and sequencing
Shingo Takano • Wei Tian • Masahide Matsuda • Tetsuya Yamamoto •Eiichi Ishikawa • Mika Kato Kaneko • Kentaro Yamazaki • Yukinari Kato •Akira Matsumura

Received: 24 November 2010 / Accepted: 17 January 2011Ó The Japan Society of Brain Tumor Pathology 2011

Abstract Isocitrate dehydrogenase 1 (IDH1) mutations
have recently been identified as early and frequent
genetic alterations in astrocytomas, oligodendrogliomas,
and oligoastrocytomas, as well as secondary glioblastomas,
whereas primary glioblastomas very rarely contain IDH1
mutations. Furthermore, a specific monoclonal antibody,IMab-1, which recognizes IDH1-R132H—the most frequent
IDH1 mutation—has been generated. IMab-1 has beenreported to react with the IDH1-R132H protein, but not
the wild-type IDH1 or the other IDH1 mutant proteins inWestern-blot analysis. However, the importance of immunohistochemistry using IMab-1 has not yet been elucidated.
In this study, we compared the findings from IMab-1
immunohistochemistry and direct DNA sequencing using 49
glioma samples. IMab-1 detected 12 out of 49 cases; however, only nine cases were found to be IDH1-R132H bydirect DNA sequencing because of a small population of

IDH1-R132H mutation-possessing tumor cells, indicatingthat IMab-1 immunohistochemistry is useful for detecting
IDH1-R132H. We conducted immunohistochemical detection in 52 cases of grade III astrocytomas. The median timeto progression (TTP) was significantly longer in the caseswith the IDH1 mutation (86.7 months) compared to thecases without the IDH1 mutation (wild type, 10.4 months)(p \ 0.01). In conclusion, the anti-IDH1-R132H-specificmonoclonal antibody IMab-1 is very useful for detecting
IDH1-R132H in immunohistochemistry, and predicting thetime to progression in grade III anaplastic astrocytomas.
Therefore, IMab-1 is likely to be useful for the diagnosis ofmutation-bearing gliomas and for determining the treatment
strategy of grade III gliomas.
Keywords IDH1  Mutation  Immunohistochemistry 
Monoclonal antibody  Glioma

Introduction
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10014-011-0023-7) contains supplementarymaterial, which is available to authorized users.
S. Takano (&)  M. Matsuda  T. Yamamoto  E. Ishikawa 
A. Matsumura
Department of Neurosurgery, Institute of Clinical Medicine,
University of Tsukuba, 1-1-1 Tennoudai,
Tsukuba, Ibaraki 305-8575, Japane-mail: shingo4@md.tsukuba.ac.jp
W. Tian  M. K. Kaneko  Y. Kato
Molecular Tumor Marker Research Team, The Oncology
Research Center, Advanced Molecular Epidemiology ResearchInstitute, 2-2-2 Iida-nishi, Yamagata 990-9585, JapanK. Yamazaki
Department of Forensic Medicine, Yamagata University Facultyof Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan

IDH1 (isocitrate dehydrogenase 1) mutations have been
identified as early and frequent genetic alterations inastrocytomas, oligodendrogliomas, and oligoastrocytomas,
as well as secondary glioblastomas [1–3]. In contrast, primary glioblastomas, as well as other systemic cancers, very
rarely contain IDH1 mutations. The IDH1 mutations are
remarkably specific to a single codon in the conserved andfunctionally important Arg132 residue in IDH1 [4].
A monoclonal antibody, IMab-1, which is specific for
the IDH1-R132H has recently been developed [5]. IMab-1
reacted with the IDH1-R132H peptide, but not with thewild-type IDH1 (IDH-wt) peptide in ELISA. In Westernblot analysis, IMab-1 reacted only with the IDH1-R132H
protein, not the IDH1-wt protein or the other IDH1

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mutants. IMab-1 could be useful for the diagnosis andbiological evaluation of mutation-bearing gliomas.
In this study, the usefulness of IMab-1 in the immunohistochemical analysis of a large number of human glioma
samples was investigated in comparison with the direct
DNA sequencing method. Furthermore, we demonstrated
the clinical significance of IMab-1 as a prognostic factor ingrade III glioma.

Materials and methods
Patients
Eighty-four consecutive patients who underwent primary
surgery between 1994 and April 2010 at Tsukuba University Hospital were included in this study. The meanpatient age at the time of the primary surgery was
47.8 ± 16.7 years (range 2–83). Pathological grading was
performed according to the WHO classification. The
tumors comprised 26 grade IV (22 glioblastomas, 3 glioblastomas with oligodendroglioma components, and 1
primitive neuroectodermal tumor), 52 grade III (24 anaplastic astrocytomas, and 9 anaplastic oligodendrogliomas
and 19 anaplastic oligoastrocytomas), 4 grade II (4 diffuse
astrocytomas) and 2 grade I (2 pilocytic astrocytomas). The
samples from glioblastomas include 25 primary tumors andone secondary tumor. Primary glioblastomas develop very
rapidly after a short clinical history, without clinical or
histological evidence of a pre-existing, less malignant
precursor lesion. Secondary glioblastoma was categorized
as WHO grade IV on the basis of histologic criteria but had
developed slowly through progression from low-grade
diffuse astrocytoma (WHO grade II) or anaplastic astrocytoma (WHO grade III) [6].
All cases underwent operation and achieved maximal
resection. Postoperative therapies were uniform depending
upon the histological findings. For grade IV and grade III
tumors, the patients received 54–60 Gy radiation therapy
followed by ACNU and temozolomide based chemotherapy. For grade II, the patients underwent no further treatment after surgery except for re-resection and radiation
therapy after recurrence.
Informed consent was obtained from each patient or thepatient’s caretaker to obtain samples and perform thesubsequent data analysis.
Sample preparation
The sample was removed during the surgery and the mostviable part of the tumor that was devoid of macroscopicallyevident necrosis was taken as the specimen. The specimen

123

was divided into two. One was fixed in 10% formalin andthe other was frozen for subsequent analysis.
Immunohistochemical analysis
IDH1-R132H protein expression was determined immunohistochemically in paraffin-embedded tumor specimens,
as described previously. Briefly, histologic sections, 5 lmin thickness, were deparaffinized in xylene, rehydrated, andheated at 100°C in citrate buffer (pH 6.0) for 5 min. Sections were incubated with the monoclonal IMab-1 antibody(kindly donated by Dr. Darell D. Bigner, Duke University
Medical Center) that specifically recognizes IDH1-R132H,
the most common glioma-derived mutation [5], overnightat 4°C at a concentration of 5 lg/ml. The DAKO LSAB kit
was used for the post-primary antibody blocker and secondary antibody. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB) for 10 min, andcounterstained with hematoxylin.
The expression of IDH1-R132H was determined bysemiquantitatively assessing the proportion of positivelystained tumor cells. The percentage of positive cells was
rated as follows: cases with C10% cells were rated as
positive, and cases with\10% cells were rated as negative,
although there is no previous reference for this definition.
In our study, IMab-1 stained diffusely without heterogeneity in grade III and IV tumors, as shown in Figs. 1 and 2.
In a positive case, almost 90% of the tumor cells werepositive, whereas they are almost completely negative inIMab-1-negative case, and there were no cases with positivities of a few percent. However, in some grade II tumors,
IMab-1-positive percentages could be underestimated due
to low tumor cell density (Fig. 3a, b). Capper et al. [7]stated that immunoreactivity was scored positive whentumor cells showed strong cytoplasmic staining, even if
only one positive cell was detected. However, we definedcases with [10% cells as positive for accuracy.
Direct DNA sequencing of IDH1 mutations
and subcloning
Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue sections using MyghtyAmp for FFPE
(Takara) according to the manufacturer’s instructions. PCRprimers for the genomic region corresponding to IDH1
exon 4, which encodes codon R132, and the flanking
intronic sequences were as follows: human IDH1 sense(50 -AATGAGCTCTATATGCCATCACTG-30 ) and human
IDH1 antisense (50 -TTCATACCTTGCTTAATGGGTG
T-30 ). The PCR conditions were 95°C for 15 min (1 cycle),followed by 35 cycles of 94°C for 30 s, 50°C for 30 s,
72°C for 30 s, and extension at 72°C for 10 min with
HotStarTaq polymerase (Qiagen). Cycle sequencing wasBrain Tumor Pathol

subcloned into pcDNA3.1/V5-His TOPO vectors (Invitrogen), and 48 clones were sequenced to confirm the R132
mutation.
MGMT immunohistochemistry
Immunohistochemistry was carried out according to thestreptavidin–biotin peroxidase (LSAB) method (DAKOLSAB2 system, DAKO, Carpinteria, CA, USA). The
mouse monoclonal antibody (MGMT Ab-1; clone MT 3.1,
Neomarker, Westinghouse, Fremont, CA, USA) was diluted 1:20. Sections were counterstained with hematoxylin.
MGMT scoring was accompanied by the determination of
the percentage of positive nuclei from regions of maximal
nuclear staining after counting 1,000 tumor cells at 4009
magnification. Cells were counted as MGMT positive if
diffuse nuclear staining was present. Endothelial cells andperivascular lymphocytes were excluded from the positive
cell count. The cutoff value for MGMT positive cells was
20% [8–10].
Statistics
The effects of IDH1 mutation on clinical outcome wereassessed by plotting survival curves using the Kaplan–
Meier method and the log-rank test for comparison
between groups. The Cox proportional hazards model wasused to test prognostic factors in univariate and multivariate analysis. Results are expressed with relative risk andits 95% confidence interval (CI). Time to progression was
defined from the day of surgery until disease progression.
Overall survival was defined from the day of surgery until
the death of the patient. Data regarding patients who survived until the end of the observation period were censoredat their last follow-up visit. Differences were considered
statistically significant when the p values were B0.05.

Results
Immunolocalization of IDH-1-R132H detectedby IMab-1 in human gliomas
Fig. 1 IDH1 immunohistochemistry. Anaplastic astrocytoma.
a Tumor periphery at low magnification (original magnification950), b tumor periphery at high magnification (original magnification
9100), c tumor center (original magnification 9100). Please note that
IDH1 is strongly positive for tumor cell cytoplasm but negative foradjacent normal glial cells

carried out using the BigDye Terminator v1.1 cycle
sequencing kit (Applied Biosystems, Foster City, CA,
USA) with the sequencing primer (50 -CCATTATCTG
CAAAAATATC-30 ). Subsequently, the PCR products wereRepresentative cases of anaplastic astrocytoma are shown in
Fig. 1. The cytoplasm of almost all tumors was strongly
stained with IMab-1 antibody, whereas adjacent brain tissue
was not (Fig. 1). IMab-1 antibody clearly stained for every
grade of glioma, grade II diffuse astrocytoma (Fig. 2a),
grade III anaplastic astrocytoma (Fig. 2b), grade III anaplastic oligoastrocytoma (Fig. 2c), and grade IV secondary
glioblastoma (Fig. 2d). By contrast, IMab-1 antibody did
not stain any grade IV primary glioblastomas (Fig. 2e). A

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Fig. 2 IDH1
immunohistochemistry.
a Diffuse astrocytoma,b anaplastic astrocytoma,c anaplastic oligoastrocytoma,d secondary glioblastoma,e primary glioblastoma.
Original magnification 9100

summary of the immunohistochemical detection of IDH1
mutation (R132H) in gliomas is shown in Table 1.
Comparison between immunohistochemistry and direct
DNA sequencing
In 49 cases, the IDH1 mutation status was determinedby both IMab-1 immunohistochemistry and direct DNA
sequencing. As shown in Table 2, three cases (two grade II
astrocytomas and one grade III anaplastic astrocytoma) thatwere determined to be wild type by direct DNA sequencing
were immunohistochemically positive for the IDH1-R132H
mutation using the IMab-1 antibody (Fig. 3). For those threecases, the first PCR products were subsequently subcloned,
and the IDH1 sequence was checked against 44–64 subclones per case. Consequently, all three cases included the
IDH1-R132H mutant sequence (9.4–16%), indicating that
not all IDH1 mutations were detected by routine direct DNA
sequencing. These results show that the IMab-1 antibody is123

more sensitive than direct DNA sequencing, and was able toidentify the IDH1-R132H mutation. Among the 49 cases, allwild-type cases and only one IDH1 mutation (R132S) were
not detectable using the IMab-1 antibody, indicating that
IMab-1 is specific for the IDH1-R132H mutation. A previous report has also shown that IMab-1 is specific against the
IDH1-R132H mutation, and does not react with the other
IDH1 mutations, such as R132C, R132L, R132S, and
R132G, in Western-blot analysis [5]. The IMab-1 antibody
appears to be superior to routine direct DNA sequencing in
the detection of IDH1-R132H mutation.
Clinical significance of IMab-1 immunohistochemistry
for grade III gliomas
Based on the superior detection ability of the IMab-1 antibody, we performed immunostaining with the IMab-1 antibody in a large number of grade III gliomas. Fifty-two casesof grade III gliomas (24 anaplastic astrocytomas and 28

Brain Tumor Pathol
Fig. 3 Four cases of wild-type
IDH1 with routine sequencing
for R132. a Oligodendroglioma,b diffuse astrocytoma,c anaplastic astrocytoma thatwas IDH1 positive withimmunohistochemistry,d anaplastic oligoastrocytoma
that wasimmunohistochemicallynegative for IDH1 with R132S
mutation. Original
magnification 9100

Table 1 Summary of immunohistochemical detection of IDH1
mutation (R132H) in gliomas
WHO grade

Pathology

Immunohistochemistry

Grade IV

GBM

Positive

2

Negative24

n 26

Percent (%)
7.7

Grade IIIAA

Positive

26

50.0

n 52

AOA

Negative

26

DA

Positive

4100

Pilocytic

Negative
Positive000

Negative

2

AO
Grade IIn4
Grade In2

GBM glioblastoma, AA anaplastic astrocytoma, AOA anaplastic oligoastrocytoma, AO anaplastic oligodendroglioma, DA diffuse astrocytoma, Pilocytic pilocytic astrocytoma

anaplastic oligoastrocytomas/anaplastic oligodendrogliomas) were immunostained and evaluated for clinicalparameters, time to progression (TTP) and overall survival.
Univariate analysis showed that the significant prognostic
factors were preoperative Karnofsky Performance Scale(KPS) score, frontal lobe involvement and IDH1 mutation for
TTP, and preoperative KPS for OS (Table 3). Kaplan–Meiercurves with and without IDH1 mutation in grade III astrocytomas are shown in Fig. 4. In grade III astrocytomas,
the median TTP was significantly longer in the cases with the
IDH1 mutation (86.7 months) compared to those without the
IDH1 mutation (wild type, 10.4 months) (p \ 0.01). TTP
and OS were not significant under other settings (anaplastic

oligoastrocytoma/anaplastic oligodendroglioma: Fig. 5; andall grade III gliomas: Fig. 6) between the IDH1 mutant andwild types. A Cox proportional hazards model in multivariate
analysis revealed that the IDH1 mutation, age\50 years andpreoperative KPS [80 were independent factors for longer
TTP and preoperative KPS for OS (Table 4).
Because the presence of IDH1 mutations was correlatedwith MGMT promoter methylation in anaplastic oligodendroglial tumors [11], and MGMT low immunoreactivity has been associated with better progression-freesurvival in patients with glioblastoma [10], we also evaluated MGMT immunoreactivity in this study. Fifty-one of
the 52 cases of grade III gliomas were available for thisanalysis. Sixteen cases were MGMT positive and 35 cases
MGMT negative. Among the 26 IDH1 mutant cases,
MGMT was positive in 6 cases and negative in 20 cases.
Among 25 IDH1 wild-type cases, MGMT was positive in
10 cases and negative in 15 cases. There was no significant
correlation between IDH1 mutation and MGMT immunoreactivity in all grade III gliomas. Also, MGMT immunoreactivity was not associated with progression and overall
survival in patients with anaplastic astrocytoma (Tables 3,4) and grade III gliomas (data not shown).

Discussion
We demonstrated the usefulness of the immunohistochemical detection of IDH1-R132H compared to routine direct123Brain Tumor Pathol
Table 2 Summary of IDH
status withimmunohistochemistry anddirect sequencing in gliomas

WHO grade

Pathology IHC

Sequence

Match (%) Subcloning

Match (%)

Grade IV GBM

Positive 2

R132H

2

100

100

n 26

Negative 24

Wild type 24

100

100

Positive 65
R132H
Wild type 1

83.3

Negative 11

R132S
1
Wild type 10

90.9

100

Positive 4

R132H

50

100

Grade III AA
n 17

AO
AOA

Grade II DA
n4100
Mutant2

Wild type 2

Mutant1

2

Negative 0
Grade I Pilocytic Positive 0
n2

Negative 2

Table 3 Predictors of time to progression and overall survival in thepatients with anaplastic astrocytoma
VariablesnTime to progression

Overall survival

Median (mo)Median (mo)p value

a

R132H

0

Wild type

2

100

100

AA TTP p<0.01

1.8

p value

\50

13

38.3

[50

11

46.9

Male

11

24.3

Female

13

48.8

71.9
0.674

77.7

0.552

75.0

0.527

Survival

MutantAge (years)

.6
.4

Sex
73.5

.2
0.533

Wild

Preoperative KPS

0

[80

8

38.6

\80

16

34.6

56.2
0.046

62.0

0.308

75.1

0

20

40

0.019

60

80

100

120

140

120

140

Time (month)

Total resection
5

60.0

No
19 38.7
Frontal involvement
Yes

15

52.0

No

9

22.2

b

85.3
0.914

0.020

39.6

0.009

23.2

0.806

IDH1 mutation
Yes

15

52.1

No

9

10.4

86.7
0.449

MGMT expression
\20%

18

42.1

[20%

6

16.0

AA OS ns1

Mutant.8

83.4

Survival

Yes

.6
.4
.2

Wild

77.7
0.854

25.5

0.718

KPS Karnofsky performance status

0
0

20

40

60

80

100

Time (month)

DNA sequencing in glioma specimens. We confirmed the
IDH1-R132H mutation after subcloning in three caseswith the wild type using routine direct DNA sequencing.
By contrast, immunohistochemical detection with the123

Fig. 4 Kaplan–Meier curve with and without IDH1 mutation inanaplastic astrocytomas. a Time to progression, b overall survival.
TTP is significantly (p \ 0.01) longer in anaplastic astrocytomas with
IDH1 mutation than in those without IDH1 mutation

Brain Tumor Pathola

AO/AOA1a

TTP ns.8.8Mutant.6

Survival

Survival

Grade III TTP ns

1

.4
.2

Mutant

.6
.4
.2

Wild
0

Wild

0

0

20

40

60

80

100

120

0

20

40

Time (month)

b

80

b

100

120

140

120

140

Grade III OS ns

1

Mutant.8

Mutant.8.6

Survival

Survival

OS ns

AO/AOA

1

60

Time (month)

.4
.2

.6
.4

Wild

.2

Wild
0

0
0

20

40

60

80

100

120

140

Time (month)Fig. 5 Kaplan–Meier curve with and without IDH1 mutation inanaplastic oligodendroglioma and anaplastic oligoastrocytomas.
a Time to progression, b overall survival

IDH1-R132H-specific monoclonal antibody, IMab-1, waspositive for these three cases. The reason for this discrepancy might be that direct DNA sequencing of highly contaminated tumor samples could not detect the IDH1-R132H
mutation, or that the tumor cell contents in three samples
were very small. To solve these problems, we should try
several approaches: (1) tumor cell-rich areas should behistopathologically identified and used for DNA sequencing
by microdissection; (2) double COLD PCR combined withhigh-resolution melting could be applied [12], resulting in*100-fold more sensitivity than Sanger sequencing; (3)frozen specimens could be used for mutation analysis. Furthermore, IMab-1 can only detect IDH1-R132H mutation,
whereas there are other IDH mutations, including IDH1R132C, IDH1-R132L, IDH1-R132S, IDH1-R132G, IDH2R172K, IDH2-R172M, and IDH2-R172G [3]. Therefore,
novel antibodies that recognize the other IDH1 mutants or
IDH2 mutants should be developed to cover all IDH mutations in immunohistochemistry.

0

20

40

60

80

100

Time (month)Fig. 6 Kaplan–Meier curve with and without IDH1 mutation in all
grade III gliomas. a Time to progression, b overall survival

Three distinct forms of isocitrate dehydrogenase (IDH1,
IDH2, and IDH3) have been identified. In contrast to other
IDHs in the mitochondria, IDH1 is present in the cytosol,and its biochemical role is not yet fully understood [2]. IDH1
mutations were found to result in the ability of the enzyme
to catalyze the reduced NADP-dependent reduction ofa-ketoglutarate to R(-)-2-hydroxyglutarate (2-HG) [13].
Reduction of a-ketoglutarate by 2-HG or mutant IDH1
results in a lower level of prolyl hydroxylases and promotes
the accumulation of hypoxia-inducible factor (HIF) 1a.
HIF-1a levels were greater in human gliomas harboring an
IDH1 mutation than in tumors without the mutation. Thus,
IDH1 appears to function as a tumor suppressor that, whenmutationally inactivated, contributes to tumorigenesis inpart through induction of the HIF-1 pathway. HIF-1a
expression is also a prognostic factor in glioma patients [14].
The link between IDH1 and HIF-1a highlights an emerging
theme in which mutationally altered metabolic enzymes arethought to contribute to tumor growth by stimulating the
HIF-1a pathway and tumor angiogenesis [15].

123Brain Tumor Pathol
Table 4 Multivariate analysis of factors associated with survival
VariableTime to progression
Hazard ratio (95% CI)

Overall survival
p value

Hazard ratio (95% CI)

p value

IDH1 (mutation vs. wild type)0.005 (0.000–0.141)

0.002

0.163 (0.022–1.234)

0.079

MGMT (\20 vs. C20%)

22.158 (0.760–646.126)

0.072

3.535 (0.209–59.924)

0.382

Age (\50 vs. C50 years)

15.068 (1.580–143.695)

0.018

1.804 (0.390–8.347)

0.45

Sex (female vs. male)

0.210 (0.029–1.518)

0.122

1.398 (0.296–6.606)

0.672

Frontal involvement (yes vs. no)0.129 (0.014–1.234)

0.076

0.878 (0.164–4.706)

0.88

KPS (80–100 vs. 10–70)

0.042 (0.004–0.496)

0.012

0.067 (0.006–0.721)

0.026

Total resection (yes vs. no)0.644 (0.036–11.667)

0.766

1.273 (0.167–9.682)

0.816

Furthermore, IMab-1 immunohistochemistry showedclinical significance as a prognostic factor in grade III
anaplastic astrocytomas. The patients with IMab-1 immunoreactive anaplastic astrocytomas had significantly longer
progression-free survival than those who were IMab-1
negative. Clinically, the IDH1 mutation strongly correlatedwith good prognosis in patients with gliomas. Medianoverall survival in GBM patients with IDH mutation was
significantly longer than that in GBM patients with wildtype IDH1 and IDH2 [3]. Mutations of IDH1 have also
been associated with improved prognosis in patients withanaplastic astrocytomas [1, 3]. Multivariate analysis has
confirmed that IDH1 mutations are independent favorable
prognostic markers in GBMs and anaplastic gliomas after
adjustment for other genomic profiles and treatment
modalities [4]. A more recent report with anaplastic oligodendroglial tumors showed the presence of IDH1
mutations correlated with 1p/19q codeletion and MGMT
promoter methylation. IDH1 mutations and 1p/19q codeletion but not MGMT promoter methylation were independent prognostic factors [11]. These reports were basedon the finding of IDH1 mutation by direct DNA sequencing
analysis; therefore, our study is noteworthy since it highlights the utility of focusing on the immunohistochemical
detection of IDH1 mutation as a prognostic factor in grade
III gliomas.
In our study of anaplastic oligodendroglioma (n 9) andanaplastic oligoastrocytoma (n 19), there was no statisticaldifference in TTP and OS between mutant-type and wildtype IDH1 tumors. Median TTP was 47.7 months forpatients with wild-type IDH1 tumors, and this was notreached by patients with mutated IDH1 tumors. Also, themedian OS was 119 months for patients with wild-type
IDH1 tumors, and this was not reached by patients with
mutated IDH1 tumors. There seems to be a tendency forbetter survival benefits in terms of both TTP and OS with
mutated IDH1 tumors. There could be three reasons why astatistical difference was not obvious in our study. First, thefollow-up period for patients was relatively short in the AO
and AOA group (average: 36 months) compared to the123anaplastic astrocytoma group (average: 48 months). Second, patient survival data in our study, even for wild-type
IDH1 tumors, was much better than in a previous report[11] (see the table in the Electronic supplementary material). In AOA and AO with wild-type IDH1, the median
TTP was 7.8 and 47.7 months in the previous report andour study, respectively. Also, in AOA and AO with wildtype IDH1, the median OS was 14.2 and 119 months in theprevious report and our study, respectively. Third, in ourstudy, the number of cases (n 28) is still small compared tothe previous report (n 76). Longer follow-up periods andmore cases are needed to draw further conclusions.

Conclusions
An anti-IDH1-R132H-specific monoclonal antibody,IMab-1, is useful for detecting IDH1-R132H in immunohistochemistry, and predicted time to progression in grade
III anaplastic astrocytomas. Therefore, IMab-1 should be
useful for the diagnosis of mutation-bearing gliomas and in
determining the treatment strategy for grade III gliomas.
Acknowledgments We gratefully acknowledge Yoshiko Tsukada
and Makiko Miyakawa for their excellent technical assistance, andChifumi Kitanaka for his great suggestion. IMab-1 antibody was
kindly donated by Dr. Darell D. Bigner, Duke University Medical
Center. This study was supported by a grant-in-aid for scientificresearch from the Japan Society for the Promotion of Science (S. T.),and by grants provided by the Tsukuba Advanced Research Alliance,
the Japan Brain Foundation, the Japanese Foundation for Researchand Promotion of Endoscopy, the Japanese Foundation for Multidisciplinary Treatment of Cancer (S.T.), the Global COE Program for
Medical Sciences, the Japan Society for the Promotion of Science(Y. K. and M. K. K), and the Experimental and Research Center,North China Coal Medical University (W. T.).

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