J Neurooncol
DOI 10.1007/s11060-012-0837-0

LABORATORY INVESTIGATION

Immunohistochemical detection of IDH1 mutation, p53,
and internexin as prognostic factors of glial tumors
Shingo Takano • Yukinari Kato • Tetsuya Yamamoto • Mika Kato Kaneko •Eiichi Ishikawa • Yuta Tsujimoto • Masahide Matsuda • Kei Nakai •Ryo Yanagiya • Shunpei Morita • Koji Tsuboi • Akira Matsumura

Received: 5 September 2011 / Accepted: 20 February 2012
Ó Springer Science+Business Media, LLC. 2012

Abstract Isocitrate dehydrogenase 1 (IDH1) mutations,which are early and frequent genetic alterations in astrocytomas, oligodendrogliomas, oligoastrocytomas, andsecondary glioblastomas, are specific to arginine 132(R132). Recently, we established monoclonal antibodies(mAbs) against IDH1 mutations: anti-IDH1-R132H andanti-IDH1-R132S. However, the importance of immunohistochemistry using the combination of those mAbs has
not been elucidated. For this study, 164 cases of glioma
were evaluated immunohistochemically for IDH1 mutations (R132H and R132S) using anti-IDH1 mAbs (HMab-1
and SMab-1). IDH1 mutation was detected, respectively, in
9.7%, 63.6%, 51.7%, and 77.8% of primary grade IV,
secondary grade IV, grade III, and grade II gliomas. For
each grade of glioma, prognostic factors for progressionfree survival and overall survival were evaluated using
clinical and pathological parameters in addition to IDH1
immunohistochemistry. IDH1 mutation, p53 overexpression, and internexin expression, as evaluated using immunohistochemistry with clinical parameters such as degree

Shingo Takano and Yukinari Kato contributed equally to this work.
S. Takano (&)  T. Yamamoto  E. Ishikawa  M. Matsuda 
K. Nakai  K. Tsuboi  A. Matsumura
Department of Neurological Surgery, Institute of Clinical
Medicine, University of Tsukuba, 1-1-1 Tennoudai,
305-8575, Ibaraki, Tsukuba, Japane-mail: shingo4@md.tsukuba.ac.jp
Y. Kato (&)  M. K. Kaneko  Y. Tsujimoto  R. Yanagiya 
S. Morita
Molecular Tumor Marker Research Team, Yamagata University
Global COE Program, Yamagata University Faculty of
Medicine, 2-2-2 Iida-nishi, 990-9585 Yamagata, Japane-mail: yukinari-k@bea.hi-ho.ne.jp

of surgical removal and preoperative Karnofsky Performance Status (KPS), might be of greater prognostic significance than histological grading alone in grade III aswell as IDH1 mutation in grade IV gliomas.
Keywords IDH1  p53  Internexin  Mutation 
Immunohistochemistry  Monoclonal antibody  Glioma

Introduction
Isocitrate dehydrogenase 1 (IDH1) and analogous IDH2
mutations, which were identified as early and frequent
genetic alterations (IDH1: 50–93%; IDH2: 3–5%) in
astrocytomas, oligodendrogliomas, and oligoastrocytomas,
as well as in secondary glioblastomas, might be the initiating events in these glioma subtypes [1–3]. In contrast,
primary glioblastomas and other systemic cancers rarely
contain IDH1 mutations. The IDH mutations are remarkably specific to a single codon in the conserved and functionally important arginine 132 residue (R132) in IDH1
and R172 in IDH2. The IDH1 mutations were found to givethe enzyme the ability to catalyze the reduced nicotinamide
adenine dinucleotide phosphate (NADP)-dependent
reduction of a-ketoglutarate to R(-)-2-hydroxyglutarate(2-HG) [4]. Results of the initial study demonstrated that
reduction of a-ketoglutarate by 2-HG or mutant IDH results
in a lower level of prolyl hydroxylases and promotes
accumulation of hypoxia-inducible factor (HIF)-1a [5].
Although HIF-1a is upregulated in a subset of gliomas
in vivo, activation of the HIF-1a pathway is not regulated
primarily by IDH1 mutation in vitro [6] or in vivo [7].
Progression of IDH1 mutant glioma might be related toalternative mechanisms such as excess accumulation of
2-HG [4].

123J Neurooncol

To date, three monoclonal antibodies against IDH1
mutations have been reported [8–11]. In this study, we
newly established an anti-IDH1-R132H-specific monoclonal antibody, HMab-1, which is expected to be extremely
useful in immunohistochemistry. This study was conducted
to evaluate the prognostic relevance of this IDH1 mutationassessed using two antibodies (HMab-1 and SMab-1)
associated with other immunohistochemically detectablefactors such as p53, internexin (INA) [12–15], and O6methylguanine-DNA methyltransferase (MGMT) expression in a large consecutive series of low-grade and highgrade gliomas.

resection and radiation therapy after recurrence. Informed
consent was obtained from each patient or the patient’s
carer for obtaining samples and subsequent data analysis.
Sample preparation
The sample was removed during surgery, and the mostviable part of the tumor that was devoid of macroscopicallyevident necrosis was taken as the specimen. The specimen
was divided into two. One was fixed in 10% formalin, andthe other was frozen for subsequent analysis.
Hybridoma production

Patients and methods
Patients
One hundred sixty-four consecutive patients who underwent primary surgery at Tsukuba University Hospital(grade II between 1994 and 2004, grade III between 1994
and 2010, and grade IV between 2008 and 2010) were
included in this study. Mean patient age at time of primary
surgery was 48.6 ± 14.3 years (range 18–83 years). Pathological grading was performed according to the World
Health Organization (WHO) classification. The tumors
comprised 52 grade IV (41 primary glioblastomas and 11
secondary glioblastomas), 66 grade III (32 anaplastic
astrocytomas, 10 anaplastic oligodendrogliomas, and 24
anaplastic oligoastrocytomas), and 46 grade II (42 diffuse
astrocytomas and 4 oligodendrogliomas). The pathologicalreview was diagnosed in our institution by three pathologists and two neurosurgeons as a routine study. Pathologically difficult cases were sent to Brain Tumor ReferenceCenter (Dr. Yoichi Nakazato; Neuropathological Section of
Gunma University) to decide a final diagnosis. Secondary
glioblastomas were categorized as WHO grade IV on thebasis of histologic criteria, but had been categorized as
WHO grade II or III at least 1 year earlier. For patientswith secondary glioblastomas, survival was calculated
from date of secondary diagnosis [1]. All cases underwent
operation and achieved maximal resection without new
permanent neurological deficits. Extent of surgery assessment was based on postoperative magnetic resonanceimaging (MRI) within 72 h after surgery. The tumoral
lesion (T1 gadolinium-enhanced lesion for grade IV and
T1 low-intensity lesion for grade II and III) was totally
removed on postoperative MRI. 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-123

BALB/c mice (CLEA Japan Inc., Tokyo, Japan) were
immunized by intraperitoneal (i.p.) injection of 100 lg synthetic peptide CKPIIIGHHAYGD (IDH1-R132H peptide;
Operon Biotechnologies, K.K., Tokyo, Japan), corresponding
to amino acids 126–137 of human IDH1-R132H plus N-terminus cysteine conjugated with KLH together with Imject
Alum (Thermo Scientific Inc., Rockford, IL). One week later,
secondary i.p. immunization of 30 lg IDH1-R132H peptide
was performed. After several additional immunizations of
30 lg IDH1-R132H peptide, a booster injection was given i.p.
2 days before spleen cells were harvested. The spleen cells
were fused with mouse myeloma P3U1 cells (American Type
Culture Collection, Manassas, VA) using Sendai virus(hemagglutinating virus of Japan, HVJ) envelope: GenomONECF (Ishihara Sangyo Kaisha, Ltd., Osaka, Japan) according to
the manufacturer’s instructions. The hybridomas were grownin Roswell Park Memorial Institute (RPMI) medium withhypoxanthine, aminopterin, and thymidine selection medium
supplement (Invitrogen Corp.). The culture supernatants werescreened using enzyme-linked immunosorbent assay (ELISA)
for binding to the IDH1-R132H peptide and the IDH1 wild
type (IDH1-WT).
Enzyme-linked immunosorbent assay (ELISA)
Synthetic peptides corresponding to amino acids 126–137
of the human IDH1: IDH1-WT (KPIIIGRHAYGD), IDH1R132H (KPIIIGHHAYGD), IDH1-R132C (KPIIIGCH
AYGD), IDH1-R132S (KPIIIGSHAYGD), IDH1-R132G(KPIIIGGHAYGD), and IDH1-R132L (KPIIIGLHAYGD)
were immobilized, respectively, on Maxisorp 96-wellimmunoplates (Thermo Fisher Scientific Inc., Waltham,MA) at 1 lg/ml for 30 min. After blocking with SuperBlock T20 (PBS) blocking buffer (Thermo Fisher ScientificInc.), the plates were incubated with 1 lg/ml of primary
antibodies, followed by 1:1,000 diluted peroxidase-conjugated anti-mouse IgG (Dako, Glostrup, Denmark). The
enzymatic reaction was conducted with a substrate solution
containing 1-Step Ultra TMB-ELISA (Thermo Fisher

J Neurooncol

Scientific Inc.). The optical density was measured at
655 nm with a Benchmark microplate reader (Bio-Rad
Laboratories Inc., Philadelphia, PA). These reactions were
performed with a volume of 50 ll at 37°C.
Immunohistochemical analysis of IDH1
IDH1-R132H, IDH1-R132S, and IDH1 wild type (WT)
protein expression was determined immunohistochemicallyin paraffin-embedded tumor specimens, as described previously [11, 16]. Anti-IDH1-R132H (HMab-1), anti-IDH1R132S (SMab-1), and anti-IDH1 (RMab-3), which were
established in this study, are now commercially available
from Medical & Biological Laboratories Co., Ltd. (MBL;
Nagoya, Japan). Expression of IDH1 was determined bysemiquantitatively assessing the proportion of positively
stained tumor cells. We defined cases with C10% cells as
positive, and cases with\10% cells were rated as negative,
although there is no previous reference for the definition. In
our study, anti-IDH1 antibodies stained diffusely without
heterogeneity in grade III and IV tumors as shown in
Fig. 1. In positive cases almost 90% of tumor cells werepositive, whereas cases negative for anti-IDH1 antibodies
were almost completely negative, and no cases with a few
percent positivity were found. However, in some grade IItumors, positive percentages for anti-IDH1 antibodies
could be underestimated due to low tumor cell density.
Cases with C10% cells were rated as positive.

Immunohistochemical analyses of MGMT, MIB-1,
p53, VEGF, and von Willebrand factor
Immunohistochemistry was carried out according to thestreptavidin–biotin–peroxidase method (Dako LSAB2
system). A mouse monoclonal antibody, MGMT Ab-1
(clone MT3.1; Neomarker, Westinghouse, CA) at dilutionof 1:20, a monoclonal MIB-1 antibody (Immunotech) at
dilution of 1:100, a monoclonal anti-human von Willebrand factor (vWF) antibody (Dako) at dilution of 1:50, a
polyclonal anti-vascular endothelial growth factor (VEGF)
antibody (A20; Santa Cruz) at dilution of 1:100, and a
monoclonal anti-p53 antibody (clone DO7; Dako) at dilution of 1:50 were used as primary antibodies. Nuclei
positive for MGMT, MIB-1, and p53 were determined bycounting at least 1,000 tumor cells in a homogeneously
stained area. The percentage of positive cells was rated asfollows: cases with C10% cells were rated as positive, andcases with \10% cells were rated as negative for both
MGMT [17, 18] and p53 [19, 20]. We decided thethreshold of p53 overexpression as [10% [19, 20]. VEGF
was defined as positive with [10% of tumor cytoplasmic
staining. The number of vessels in a 2009 field (1.0 mm2)
was measured in microvessel ‘‘hot spots’’ (i.e., microscopic
areas containing the densest collections of microvessels, as
initially identified under low-power magnification) under
an Olympus microscope (AHBT3; Olympus, Tokyo,
Japan) on tissue sections stained for vWF. Vascular density
was defined by averaging the number of vessels in the threemost vascularized areas.
Immunohistochemical analysis of internexin neuronalintermediate filament protein alpha (INA)
INA immunohistochemistry was carried out on 5-lm paraffin sections of formalin-fixed tumor samples according to
the streptavidin–biotin–peroxidase method (Dako LSAB2
system) using an antibody that targeted INA (clone
2E3MOI; Novus Biologicals, Interchim, Montlucon,
France; 1:100 dilution). Positive INA results were observed
either as fibrillar, crescent-shaped or more paranuclear,
dot-like intracytoplasmic inclusions. Positively stainedneural cells were used as internal positive control, especially for negative slides. Labeling was defined as strong([10% positive cells), weak (\10% positive cells), or
negative (no positive tumor cells detected) [12].

Fig. 1 Production of a specific monoclonal antibody against IDH1R132H and IDH1 wild type. Synthetic peptides corresponding toamino acids 126–137 of the human IDH wild type and IDH1 mutants
were immobilized on 96-well plates. After blocking, the plates wereincubated with anti-IDH1 antibodies, followed by peroxidase-conjugated anti-mouse IgG. The enzymatic reaction was conducted with asubstrate solution containing TMB

Statistical analysis
Overall survival (OS) and progression-free survival (PFS)
were calculated from time of surgery until death, diseaseprogression, or last follow-up examination according to the
Kaplan–Meier method with log-rank test for comparison123

J Neurooncol

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).

Results
Production of a novel IDH1-R132H-specific antibody
For this study, we immunized mice with synthetic peptides
of IDH1-R132H mutant. After cell fusion using the Sendai
virus envelope, the wells of hybridomas, which produced
IDH1-R132H-specific antibodies, were selected in ELISA.
After limiting dilution, one clone was established: HMab-1(IgG1 subclass). We also established RMab-3 (IgG1 subclass), which reacts with both IDH1-R132H and IDH1 wild
type (IDH1-WT). To determine the specificity of HMab-1
monoclonal antibody, the reactivities against IDH1-WTand the IDH1 mutant (R132H, R132C, R132S, R132G,
R132L) peptides were investigated using ELISA. Results
showed that HMab-1 reacted with IDH1-R132H peptide
but not with IDH1-WT or other IDH1 mutant (R132C,
R132S, R132G, R132L) peptides, whereas RMab-3 reactedwith both IDH1-WT and all IDH1 mutant peptides (Fig. 1),
indicating that HMab-1 is a specific antibody against
IDH1-R132H peptide and that RMab-3 recognizes thecommon epitope of IDH1. In addition, SMab-1 showed
weak cross-reaction to R132G (Fig. 1).
IDH1 immunohistochemical analysis
HMab-1 and SMab-1 were confirmed as anti-IDH1R132H-specific and anti-IDH1-R132S-specific antibodies
in ELISA, respectively. Therefore, we next performed
immunohistochemistry of HMab-1 and SMab-1 against
IDH1-R132H-positive or IDH1-R132S-positive gliomas,
whose mutations were determined by direct sequencing[11]. Typical results are presented in Fig. 2. HMab-1
stained almost all tumor cells of IDH1-R132H-positive
glioma (AOA: anaplastic oligoastrocytoma, case 1),
although no staining was observed in IDH1-R132S-positive glioma (AOA, case 2) or IDH1-WT glioma (GBM:
glioblastoma, case 3). In fact, HMab-1 stained no endothelial cells (data not shown). Furthermore, SMab-1 stained
no tumor cells in IDH1-R132H-positive gliomas (AOA,
case 1), although SMab-1 stained IDH1-R132S-positive
gliomas (AOA, case 2). RMab-3, which recognizes common epitopes of IDH1, reacted with all glioma types(cases 1, 2, and 3), although the RMab-3 reactivity isapparently heterogeneous. These results indicate that
HMab-1 and SMab-1 are useful in respective immunohistochemical analyses for detection of IDH1-R132H and123

IDH1-R132S mutations. A summary of the immunohistochemical detection of IDH1 mutations (R132H and R132S)in gliomas is presented in Table 1. IDH1 mutation was
detected, respectively, in 9.7%, 63.6%, 57.1%, 46.6%,
75.0%, and 100% of primary grade IV, secondary grade IV, grade III anaplastic astrocytoma, grade III anaplastic oligodendroglioma/anaplastic oligoastrocytoma,
grade II diffuse astrocytoma, and grade II oligodendroglioma. The frequency of IDH1-R132H was 42.5% (62/146),
and that of IDH1-R132S was 4.8% (7/146).

Clinical significance of HMab-1 and SMab-1
immunohistochemical analyses for gliomas
A summary of other immunohistochemical detections of
p53, MGMT, and INA in the respective grades is presented
in Table 1. In patients with 66 grade III gliomas, clinicalparameters such as age, sex, preoperative KPS, tumor
location, degree of tumor removal, postoperative chemotherapy, and other pathological parameters such as INA
expression, MGMT expression, MIB-1 positivity, vWFstained vessel number, and p53 expression were evaluatedto determine progression-free and overall survival in
addition to IDH1 mutation. Results of univariate analyses
indicated that the significant prognostic factors were IDH1
mutation positivity, MIB-1 \20%, INA positivity, and p53\10% for PFS, and IDH1 mutation positivity, INA positivity, preoperative KPS C80, and MIB-1 \20% for OS(Table 2). Results of multivariate analyses showed that theindependent significant prognostic factors were IDH1
mutation positivity, total removal, and p53 \10% for PFS,
and IDH1 mutation positivity, total removal, MIB-1\20%,
preoperative KPS C80, and p53 \10% for OS. Kaplan–
Meier curves with and without these prognostic factors ingrade III gliomas are portrayed in Fig. 3. PFS was significantly longer in cases with IDH1 mutation positivity(80 months) than in those with IDH1 mutation negativity(23 months) (p = 0.001) (Fig. 3a), for total removal(84 months) versus without total removal (38 months)(Fig. 3b), for p53 \10% (80 months) versus p53 C10%
(24 months) (Fig. 3c), and for INA positivity (68 months)
versus INA negativity (14 months) (Fig. 3d). In addition,
the figure shows that OS was significantly longer in caseswith IDH1 mutation positivity (119 months) than in thosewith IDH1 mutation negativity (33 months) (Fig. 3g), forpreoperative KPS [80 (117 months) versus KPS \80(26 months) (Fig. 3h), for p53 \10% (117 months) versus
p53 C10% (56 months) (Fig. 3i), and for INA positivity(117 months) versus INA negativity (33 months) (Fig. 3j).
Taking these facts together, IDH1 mutation positivity and
p53 \10% were shown to be the most striking prognostic
factors with subsequent INA expression.

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Fig. 2 Immunohistochemical analyses by anti-IDH1 antibodies
against glioma tissues. Glioma tissues having IDH1-R132H (AOA:
anaplastic oligoastrocytoma, case 1), IDH1-R132S (AOA, case 2),

and wild type (GBM: glioblastoma, case 3) were stained with HMab1 (anti-IDH1-R132H), SMab-1 (anti-IDH1-R132S), and RMab-3(anti-IDH1-WT). Magnification 9200

Table 1 Summary of immunohistochemical analysis of each glioma grade
IDBI

p53

MGMT

INA

R132H

R132S

Positive

Positive

Positive

Positive3

1

4/41 (9.7%)

14/35 (40.0%)

7/31 (22.5%)

9/41 (21.9%)

Grade IV

Primaryn = 41

Secondaryn = 116

1

7/11 (63.6%)

5/10 (50.0%)

1/4 (25.0%)

10/11 (90.9%)

Grade IIIAAn = 3216

0

16/28 (57.1%)

12/32 (37.5%)

10/28 (35.7%)

17/30 (56.7%)

AO/AOAn = 3411

3

14/30 (46.6%)

13/33 (39.4%)

14/31 (45.1%)

23/32 (71.9%)

DA

n = 4222

224/32 (75.0%)

11/37 (29.9%)

ND

22/33 (66.7%)

Oligo

n=4

4

0

4/4 (100%)0/1 (0%)ND

4/4 (100%)

Grade IIIn 52 patients with grade IV gliomas, clinical parameters and other pathological parameters that were used in
grade III gliomas were evaluated to determine progressionfree and overall survival in addition to IDH1 mutation.
Univariate analysis showed that IDH1 mutation positivity
had a tendency as a prognostic factor for PFS and was asignificant prognostic factor for OS. Multivariate analysis
showed that independent significant prognostic factors
were IDH1 mutation positivity and sex (male predominance) for OS (Table 3A, B). Kaplan–Meier curves
showed that PFS was significantly longer in cases with
IDH1 mutation positivity (20 months) than in those with123

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Table 2 Prognostic factors of(A) grade III glioma: PFS,
(B) grade III glioma: OS

Hazard ratio

95% CI

p value

0.9375(A)
PFS univariate
Age (years)\40 vs. [40

0.971

0.466–2.022

Sex

M vs. F

0.875

0.432–1.774

0.7114KPS[80 vs. \80

0.522

0.254–1.073

0.0771

Location

Frontal vs. others

0.646

0.309–0.350

0.2451

Total removal

Yes vs. no0.412

0.144–1.175

0.0973

Pathology

AA vs. AO

1.467

0.731–2.944

0.2805Chemo

ACNU vs. TMZ

0.827

0.187–3.652

0.8017

IDH1

Pos vs. neg

0.272

0.120–0.620

0.0019INA

Pos vs. neg

0.34

0.151–0.765

0.0091MGMT

Pos vs. neg

0.864

0.358–2.086

0.7455MIB-1[20% vs. \20%2.593

1.066–6.306

0.0356

Vessel[50 vs. \50

1.25

0.520–3.007

0.615

p53
PFS multivariate

Pos vs. neg

2.312

1.097–4.873

0.0276KPS[80 vs. \80

0.94

0.342–2.579

0.9038

Total removal

Yes vs. no0.059

0.012–0.302

0.0007

Pathology

AA vs. AO

1.97

0.714–5.433

0.1901

IDH1

Pos vs. neg

0.088

0.023–0.333

0.008

INA

Pos vs. neg

0.895

0.266–3.014

0.8584MIB-1[20% vs. \20%1.612

0.444–5.849

0.468

p53

Pos vs. neg

7.037

2.504–19.778

0.0002\40 vs. [40

1.231

0.535–2.835

0.6251(B)
OS univariate
Age (years)
Sex

M vs. F

0.819

0.370–1.811

0.6216KPS[80 vs. \0.28

0.125–0.646

0.0028Location

Frontal vs. others

1.001

0.434–2.308

0.9984

Total removal

Yes vs. no0.287

0.067–1.225

0.0917

Pathology
Chemo

AA vs. AO
ACNU vs. TMZ

1.581
0.501

0.724–3.453
0.060–4.174

0.25050.5229IDH1

Pos vs. neg

0.294

0.115–0.747

0.0101INA

Pos vs. neg

0.38

0.166–0.867

0.0215MGMT

Pos vs. neg

0.717

0.240–2.147

0.5526MIB-1[20% vs. \20%3.003

1.066–8.454

0.0373Vessel[50 vs. \50

1.212

0.434–3.389

0.7134p53

Pos vs. neg

1.767

0.804–3.880

0.1563OS multivariate
KPS[80 vs. \80

0.502

0.100–0.907

0.0328

Total removal

Yes vs. no0.12

0.020–0.714

0.0197

Pathology

AA vs. AO

2.429

0.761–7.757

0.134

IDH1

Pos vs. neg

0.256

0.068–0.959

0.0432INA

Pos vs. neg

2.897

0.683–12.284

0.1488MIB-1[20% vs. \20%7.49

1.389–41.618

0.0203p53

Pos vs. neg

3.003

1.04–8.673

0.0421

IDH1 mutation negativity (9 months) (Fig. 3e). In addition, OS was significantly longer in cases with IDH1
mutation positivity (26 months) than in those with IDH1

123

mutation negativity (14 months) (Fig. 3k). IDH1 mutation
positivity was shown to be the most striking prognostic
factor in grade IV gliomas.

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Fig. 3 Kaplan–Meier curves for progression-free (a–f) and overall (g–l) survival in grade III gliomas (a–d, g–j), grade IV gliomas (e, k), andgrade II gliomas (f, l)

In patients with 46 grade II gliomas, postoperative
irradiation and tumor size [6 cm were evaluated as additional clinical parameters and VEGF expression as anadditional pathological parameter to determine progression-free and overall survival in addition to IDH1 mutation.
Univariate analysis showed that the significant prognostic
factor was tumor size \6 cm for PFS (Table 3C), and total
removal and tumor size \6 cm for OS (Table 3D). Multivariate analysis showed that significant prognostic factors
were age \40 years, total removal, and MIB-1 \2.5% for
OS. IDH1 mutation positivity was not included in any
prognostic factor, and showed no survival benefit on the
Kaplan–Meier curve (Fig. 3f, l).

Discussion
Clinically, the IDH1 mutations correlated strongly withgood prognosis in patients with gliomas, suggesting that
future clinical trials might require stratification by IDH1
mutation status [1, 2, 21]. Multivariate analyses confirmedthat IDH1 mutations are independent favorable prognostic
markers in GBMs and anaplastic gliomas after adjustment
for other genomic profiles and treatment modalities [22].

IDH1 mutations are also correlated with 1p/19q codeletionand MGMT promoter methylation in anaplastic oligodendroglial tumors [23]. Furthermore, IDH1 mutation status inanaplastic astrocytomas as well as glioblastomas was themost powerful single prognostic factor of overall patient
survival, followed by age, tumor type, and MGMT methylation status [24]. Although these reports were based on
the finding of IDH1 mutations by direct DNA sequencing
analysis, we recently reported that anti-IDH1-R132H mAb
immunohistochemistry revealed clinical significance as aprognostic factor in grade III anaplastic astrocytomas [11].
Patients with anti-IDH1-R132H-immunoreactive anaplastic astrocytomas had significantly longer progression-freesurvival than those who were anti-IDH1-R132H negative.
At present, it is recommended that all IDH1-R132Himmunonegative cases be evaluated by direct sequencing[25], because immunohistochemical approach has sensitivity of 94% because of the lack of detection of other typesof IDH1 mutations [26]. In this study, HMab-1/SMab-1positive patients had significantly longer progression-free
as well as overall survival than those who were HMab-1/
SMab-1 negative in grade III and grade IV gliomas.
Although HMab-1 alone does not reveal statistical significance for OS on multivariate analysis (hazard ratio, 0.359;

123

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Table 3 Prognostic factors of (A) grade IV glioma: PFS, (B) grade IV glioma: OS, (C) grade II glioma: PFS, and (D) grade II glioma: OS
Hazard ratio

95% CI

p value

0.415

0.141–1.218

0.1095(A)
PFS univariate
Age (years)\40 vs. [40Sex

M vs. F

0.693

0.341–1.409

0.3106KPS[80 vs. \80

1.048

0.541–2.231

0.7957

Diagnosis

Primary vs. second

1.486

0.714–3.096

0.2895

Location

Frontal vs. others

0.87

0.392–1.933

0.7325

Total removal

Yes vs. no0.472

0.136–1.645

0.2388

IDH1

Pos vs. neg

0.396

0.155–1.011

0.0527INA

Pos vs. neg

0.711

0.309–1.635

0.4223

MGMT
MIB-1

Pos vs. neg[20% vs. \20%

0.753
1.454

0.249–2.275
0.688–3.070

0.61550.3265Vessel[50 vs. \50

1.048

0.326–3.368

0.9377p53

Pos vs. neg

1.165

0.544–2.495

0.6935Age (years)\40 vs. [400.21

0.037–1.199

0.0791Sex

M vs. F

0.283

0.079–1.014

0.525

KPS

[80 vs. \80

1.413

0.488–4.091

0.5234

Diagnosis

Primary vs. second

1.226

0.410–3.667

0.7158

Total removal

Yes vs. no0.719

0.048–10.839

0.8115

IDH1

Pos vs. neg

0.173

0.032–0.934

0.0415INA

Pos vs. neg

3.882

1.155–13.046

0.282

MIB–1

[20% vs. \20%1.162

0.354–3.813

0.8047p53

Pos vs. neg

0.472

0.151–1.476

0.1969Age (years)
Sex\40 vs. [40
M vs. F

0.424
0.675

0.123–1.454
0.208–1.582

0.17210.3659KPS[80 vs. \80

0.6

0.257–1.399

0.2368Diagnosis

Primary vs. second

1.1420.476–2.737

0.7661

Location

Frontal vs. others

0.958

0.380–2.414

0.9278

Total removal

Yes vs. no0.425

0.097–1.854

0.255

IDH1

Pos vs. neg

0.239

0.079–0.729

0.0119INA

Pos vs. neg

0.7

0.274–1.791

0.4565MGMT

Pos vs. neg

1.845

0.552–6.165

0.3169

PFS multivariate(B)
OS univariate

MIB-1[20% vs. \20%

1.992

0.792–4.762

0.1469Vessel[50 vs. \50

0.286

0.076–1.077

0.0642p53

Pos vs. neg

1.269

0.524–3.070

0.5977Age (years)\40 vs. [400.169

0.015–1.897

0.1414Sex

M vs. F

0.15

0.024–0.924

0.0408KPS
Diagnosis[80 vs. \80
Primary vs. second

0.272
0.506

0.053–1.404
0.107–2.389

0.11990.3896

Total removal

Yes vs. no2.731

0.080–93.132

0.5768

IDH1

Pos vs. neg

0.061

0.005–0.795

0.0328INA

Pos vs. neg

4.37

0.818–23.375

0.0846MIB-1[20% vs. \20%1.563

0.320–7.641

0.5812p53

Pos vs. neg

0.577

0.158–2.110

0.4059OS multivariate

123

J Neurooncol
Table 3 continued
Hazard ratio

95% CI

p value

0.972

0.477–1.982

0.9376(C)
PFS univariate
Age (years)\40 vs. [40Location

Frontal vs. others

0.596

0.286–1.243

0.1677

Total removal

Yes vs. no0.453

0.173–1.182

0.1055

Radiation firstYes vs. no1.971

0.834–4.627

0.1193MIB-1[2.5% vs. \2.5%

1.27

0.585–2.754

0.5454p53

Pos vs. neg

1.114

0.507–2.444

0.7884

VEGF

Pos vs. neg

1.223

0.510–2.935

0.6516

IDH1

Pos vs. neg

0.981

0.426–2.259

0.9641INA

Pos vs. neg

0.907

0.383–2.150

0.8249Tumor size

[6 vs. \6 cm

3.47

1.450–8.303

0.0052

VesselPFS multivariate[22 vs. \22

1.014

0.428–2.402

0.9744Age (years)\40 vs. [40

0.291

0.075–1.133

0.0751

Total removal

Yes vs. no0.618

0.166–2.237

0.4728

Radiation firstYes vs. no3.837

0.805–18.202

0.0913MIB-1[2.5% vs. \2.5%

2.186

0.539–8.861

0.2732p53

Pos vs. neg

0.531

0.142–1.983

0.3459IDH1

Pos vs. neg

1.688

0.639–4.485

0.2945INA

Pos vs. neg

2.037

0.432–9.604

0.3633Tumor size

[6 vs. \6 cm

3.304

0.793–13.768

0.1007Age (years)\40 vs. [40

0.903

0.427–1.909

0.7895

Location

Frontal vs. others

0.823

0.380–1.78

0.6219Total removal

Yes vs. no0.258

0.078–0.859

0.0272

Radiation first
MIB-1

Yes vs. no[2.5% vs. \2.5%

2.197
1.555

0.890–5.423
0.679–3.559

0.0877
0.2962p53

Pos vs. neg

0.917

0.390–2.154

0.8426VEGF

Pos vs. neg

1.307

0.575–3.314

0.5729IDH1

Pos vs. neg

0.759

0.322–1.790

0.5286INA

Pos vs. neg

1.117

0.454–2.747

0.8103Tumor size

[6 vs. \6 cm

4.354

1.804–10.510

0.0011

Vessel[22 vs. \22

1.014

0.428–2.402

0.9744Age (years)\40 vs. [400.215

0.050–0.929

0.0395

Total removal

Yes vs. no0.103

0.016–0.672

0.0175

Radiation firstYes vs. no3.551

0.730–17.275

0.1163MIB–1[2.5% vs. \2.5%

8.907

1.552–51.1

0.0141p53

Pos vs. neg

0.389

0.098–1.545

0.1797

IDH1

Pos vs. neg

1.279

0.436–3.757

0.654

INA

Pos vs. neg

2.133

0.350–12.99

0.4112Tumor size

[6 vs. \6 cm

3.098

0.635–15.106

0.1619(D)
OS univariate

OS multivariate

95% CI, 0.124–1.038; p value, 0.0587), combined use of
HMab-1/SMab-1 revealed statistical significance for OS on
multivariate analysis (Table 2B; hazard ratio, 0.256; 95%

CI, 0.068–0.959; p value, 0.0432), indicating that combined use of HMab-1/SMab-1 is a powerful tool to makeprognoses of patients with grade III glioma.

123

J Neurooncol

In our study, IDH1 mutation was not a prognostic factor
in grade II gliomas. Some have reported that grade II diffuse glioma patients with mutated IDH1/2 demonstrated
better prognosis [7, 22, 27], although another report
described that IDH1 mutation was not a prognostic factor
in grade II gliomas [28, 29]. Thon et al. reported that IDH1
mutations in grade II astrocytomas are associated withunfavorable progression-free survival and prolonged postrecurrence survival [28]. Significant differences werereported for survival in a group of 77 IDH1-mutated(median OS, 150.9 months) and 23 IDH-nonmutated(median OS, 60.1 months) grade II gliomas (p = 0.01)[22]. Positive prognostic impact of IDH1 mutation wasreported on OS in a population of 49 low-grade astrocytomas, even if the analysis was performed at time of progression [27]. However, their reports did not demonstrate
the role of prognostic factors for PFS in grade II gliomas[22, 27]. A recent report showed that IDH mutations arestrongly associated with prolonged survival in low-grade
glioma (n = 271) on univariate and multivariate analyses,
although PFS was not different between IDH1 mutation
positivity and mutation negativity [30]. However, thesestudies do not include important clinical parameters such as
tumor size, which was a significant prognostic factor forboth PFS and OS on our univariate analysis and others[31]. In addition, for grade II gliomas, various strategies
including chemotherapy, radiation therapy, and othermodalities after surgery other than IDH1 mutation cancause various genetic changes in the tumor. To study therole of IDH mutation on the spontaneous growth (natural
history) of low-grade glioma, PFS was investigated in aseries of 171 patients who had no adjuvant treatment after
surgery until first progression. Spontaneous PFS did not
differ in IDH-mutated and IDH-wild-type patients [30].
Taken together, the IDH1 mutation is a controversial
prognostic factor in grade II gliomas. Further studies,particularly addressing special location [32] or uniform
treatment group, must be undertaken to determine the exact
significance of IDH1 mutation in grade II gliomas.
Increased expression of p53 most likely reflects thepresence of loss-of-function mutations in the protein [33].
Mutations of the p53 gene are most commonly found in
low-grade gliomas and younger patients [34]. They are
believed to be related to invasive and aggressive nature or
malignant astrocytomas [19]. In our study, patients with
p53 expression (C10%) had significantly shorter progression-free and overall survival in grade III gliomas. IDH
mutation positivity and p53 \10% have been shown to be
the most striking prognostic factors, although cases thatwere IDH mutation negative with p53 [10% (10 of 58
grade III gliomas) had the shortest PFS of 12.0 months.
Recently, Birner et al. [35] reported strong correlation
between IDH1 mutations and p53 expression. In our study,

123

no significant correlation was found between IDH1 mutations and p53 expression (data not shown), although p53
expression was an independent prognostic factor for PFS in
grade III gliomas. This discrepancy can be explained by
the difference of p53-positive criteria. Our criteria arequantitative ([10% nuclear staining), whereas their criteria
were semiquantitative, merely reflecting whether it wasstrong or not.
Although recent studies emphasized the importance of
immunostaining as a screening procedure to identify
patients with mutant p53 DNA alleles, immunostaining canalso reveal an expanded spectrum of diseases because ofoverexpression of nonmutant or wild-type p53 [36]. The
existence of a subset of astrocytic tumors overexpressing
p53 protein in the absence of detectable p53 mutations has
been demonstrated [20]. Overexpression of p53 protein
was not always associated with point mutations in conserved exons of the p53 gene in astrocytic tumors. Evaluation of p53 protein expression as a continuous variable
rather than as a binary variable also demonstrated higher
levels of statistical significance for PFS, suggesting thatoverexpression of p53 protein is important in the natural
growth of these aggressive tumors [20]. Use of p53
immunostaining as a prognostic indicator, in contrast to
mutational DNA analyses, might be a useful adjunct for
identifying patients at higher risk of treatment failure. Thisfinding implies a distinct role of wild-type p53 expression
in the natural growth of astrocytomas. Taken together, ourfinding of p53 overexpression as well as IDH1 mutations as
having prognostic significance is valuable in terms of thenatural growth of astrocytic tumors.
Close functional and genetic relation between IDH1
mutations and 1p/19q codeletion has been reported intensively [37]. In practice, the most widely available techniques to detect 1p/19q codeletion include fluorescentin situ hybridization (FISH), loss of heterozygosity (LOH),
multiplex ligation-dependent probe amplification (MLPA),
and comparative genomic hybridization (CGH) array. All
have their respective limitations: contamination with normal cells might impair genetic analysis; LOH and FISH are
inadequate to distinguish whole 1p and whole 19q loss
from partial 1p loss frequently associated with 1p and 19q
loss. These prognoses are radically different, and theamount of available tissue, in cases of biopsy, might beinsufficient for CGH array. Results of the Ducray study
suggest that INA expression can serve as a surrogate
marker for 1p/19q-codeleted tumors [12]. Their data indicate that absence of INA expression in an oligodendroglial
tumor makes 1p/19q deletion very unlikely (1/48: 2%). In
contrast, when [10% of cells express INA, there is [80%chance of finding 1p/19q codeletion in the tumor. Recent
retrospective analysis revealed that INA expression isrelated more closely to CGH than to FISH. Furthermore,J Neurooncol

INA expression is related closely to MGMT promoter
methylation and IDH1/2 mutations. INA expression is also
prognostic with IDH1-nonmutated glioma [15]. In ourstudy, INA expression was shown to be a significant
prognostic factor for both PFS and OS on univariate
analysis, with significant longer survival on the Kaplan–
Meier curve in grade III gliomas. However, the exact
prognostic and predictive significance of INA status inpatients with grade IV and grade II gliomas was notobserved. This must be determined based on results offuture studies, as results of other studies have suggested[13, 14].
We used an immunohistochemical approach against
MGMT expression in this study. A recent report described
only weak to moderate correlation between MGMT
immunoreactivity and MGMT promoter methylation [38].
However, Christmann et al. [18] found agreement between
immunoreactivity and MGMT activity ([30 fmol/mg protein) in 75% of cases. Correlation between promoter
methylation and immunoreactivity was only slightly lower(in 72.5% of cases for MSP-P1 and 62.5% of cases for
MSP-P2). The combination of several methods mightprovide more useful prognostic data. Consequently, it wasrecently demonstrated that the combination of MGMT
promoter methylation and negative MGMT expression was
significantly associated with increased overall survival,
although a correlation between promoter methylation andimmunostaining was observed in only 50% of samples[39]. Immunohistochemical approaches for MGMT might
be useful in limited circumstances.
IDH mutation status is now regarded as an independent
positive predictive factor for glioblastoma [22] and WHO
grade II and III astrocytomas [21, 30]. In our study, IDH1
mutation status was confirmed as a strong prognostic factor
in grade III and grade IV with longer overall survival aswell as longer progression-free survival than their wildtype counterparts, although IDH1 mutation status was not a
prognostic factor in grade II gliomas. The most striking
finding evaluated by either immunohistochemistry or direct
sequencing might be the greater prognostic significancethan that of histological grading [25]. Immunohistochemical detection of IDH mutations combined with p53 and
INA is expected to be a promising technique for diagnosis
and tumor classification in gliomagenesis.
In summary, these striking findings of IDH1 mutations(R132H and R132S) and p53 mutation, evaluated using
immunohistochemistry with clinical parameters such as
degree of surgical removal and preoperative KPS, might beof greater prognostic use for grade III gliomas than histological grading alone. In addition, IDH1 mutations (R132H
and R132S) might be of great prognostic use as factors toevaluate grade IV gliomas. Because of the high mutation
frequency, mutated IDH1 screened by the combination

HMab-1/SMab-1 can potentially form a good target and/or
biomarkers for new treatments.
Acknowledgments We thank Yoshiko Tsukada, Makiko Miyakawa, Imi Saito, and Junko Aita for their excellent technical assistance.
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan (S.T., Y.K., M.K.K.), Japan Brain Foundation (S.T.), The Japanese Foundation for research and promotion ofendoscopy (S.T.), Japanese Foundation for Multidisciplinary Treatment of Cancer (S.T.), Mitsubishi Pharma Research Foundation(Y.K., M.K.K.), Children Cancer Association of Japan (Y.K.), Intelligent Cosmos Academic Foundation (Y.K.), and Office for Gender
Equality of Yamagata University (M.K.K.).

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