Brain Tumor Pathol
DOI 10.1007/s10014-015-0222-8

ORIGINAL ARTICLE

Evaluation of IDH1 status in diffusely infiltrating gliomas
by immunohistochemistry using anti-mutant and wild type IDH1
antibodies
Hayato Ikota1 • Sumihito Nobusawa1 • Hideo Arai2 • Yukinari Kato3 •Keisuke Ishizawa4 • Takanori Hirose5 • Hideaki Yokoo1

Received: 18 October 2014 / Accepted: 13 May 2015
! The Japan Society of Brain Tumor Pathology 2015

Abstract Glioma cells with the isocitrate dehydrogenase(IDH) 1 G395A mutation are strongly immunopositive for
mIDH1R132H, an antibody against mutant IDH1R132H(clone H09). However, we encountered some gliomas
which were ambiguously positive for mIDH1R132H despite
having the IDH1 G395A mutation. The aim of this study
was to establish an evaluation procedure of IDH1 status byimmunohistochemistry. Forty-three diffusely infiltrating
gliomas were studied, and four of eight anaplastic
oligoastrocytomas with the IDH1 G395A mutation weremodestly or weakly positive for both the mIDH1R132H andan antibody against wild type IDH1, RcMab-1. Based on
our staining results, the IDH1 expression of both wild andmutated types seemed to be codominant and also to beevenly suppressed under a certain condition. We propose aprocedure for determining IDH1 status. If a glioma is
weakly positive for mIDH1R132H, immunohistochemistryfor RcMab-1 should be performed. If the tumor cells arestrongly positive for RcMab-1, the IDH1 G395A mutation
is judged to be absent on the grounds that IDH1 expression& Hayato Ikota
ikota@gunma-u.ac.jp
1

Department of Human Pathology, Gunma University
Graduate School of Medicine, 3-39-22 Showa-machi,
Maebashi, Gunma 371-8511, Japan2

Department of Diagnostic Pathology, Gunma University
Hospital, Maebashi, Japan3

Department of Regional Innovation, Tohoku University
Graduate School of Medicine, Sendai, Japan

4

Department of Pathology, Saitama Medical University,
Saitama, Japan

5

Department of Pathology for Regional Communication, Kobe
University, Kobe, Japanis not suppressed. If the tumor cells are weakly positive forboth mIDH1R132H and RcMab-1, then a conclusion should
be made after DNA sequencing. This procedure is usefulfor practical evaluation of IDH1 status.
Keywords

Glioma ! IDH1 ! Immunohistochemistry

Introduction
Isocitrate dehydrogenase (IDH) 1 or IDH2 genes are mutated in 50–80 % of World Health Organization (WHO)grade II and III diffuse gliomas, and secondary glioblastomas, suggesting that the IDH1/2 mutation is an early eventin the development of diffuse gliomas [1]. IDH1 is located in
the cytoplasm and peroxisomes, and the main function is tocatalyze oxidative decarboxylation of isocitrate into a-ketoglutarate (aKG) [2]. Mutant IDH1 acquires the ability to
reduce aKG into D-2-hydroxyglutarate (D-2HG) in an
NADPH-dependent manner [3]. It has been suggested thatmutant IDH1 contributes to gliomagenesis by D-2HG production. One hypothesis is that D-2HG inhibits prolyl hydroxylase-mediated degradation of HIF-1a, resulting in
induction of vascular endothelial growth factor expression[4]. Another possibility is that marked elevation of D-2HG
results in CpG hypermethylation at a large number of loci,which results in what is termed a glioma-CpG island
methylator phenotype (G-CIMP) [5, 6]. G-CIMP is considered to have down-regulation of target gene expression,some of which may act as tumor suppressors. The mostfrequent type of IDH mutation is G395A, causing the aminoacid substitution of arginine for histidine (R132H) (89.7 %),whereas IDH2 mutations are rare (4.4 %) [7].
In 2009, two anti-IDH1R132H monoclonal antibodies
were reported (mIDH1R132H, clone H09 [8]; clone IMab-1

123

Brain Tumor Pathol[9]), and detection of IDH1 mutations by immunohistochemistry was made possible. Glioma cells with the IDH1
G395A mutation show strong cytoplasmic and weaker
nuclear staining for mIDH1R132H [8, 10]. However, wehave encountered some anaplastic oligoastrocytoma cases
which showed ambiguous positive staining for
mIDH1R132H throughout the tissue despite harboring the
IDH1 G395A mutation. This discrepancy between immunohistochemical and DNA sequencing results hampers
precise diagnosis. Recently, molecular targeting therapy
and immunotherapy against IDH1/2 mutations have beendeveloped [11–13]; therefore, the strict confirmation of
IDH status is becoming more important.
As IDH1/2 mutations in gliomas are usually restricted to
one allele, wild type IDH1/2 is expressed even in IDH-mutated tumors. We hypothesized that the expression of both
IDH1 mutant and wild types may be suppressed by an unknown mechanism. The aim of this study was to investigate
the relation between mutant and wild type IDH1 expression in
diffusely infiltrating gliomas and to propose an evaluation
procedure of IDH1 status by immunohistochemistry.

Materials and methods
Glioma cases
A total of 43 diffusely infiltrating glioma cases were included in this study (Table 1). Forty-one tissue specimens
were obtained from the pathology archives of Gunma
University Hospital and Gunma University Graduate
School of Medicine. Two tissue specimens were provided
by other institutions and have been reported elsewhere [14,15]. The histological types were as follows: 4 diffuse astrocytomas, 4 anaplastic astrocytomas, 1 glioneuronal tumor with neuropil-like islands (GTNI) [14], 5
glioblastomas, 1 glioblastoma with oligodendroglioma
component, 4 oligodendroglioma, 1 oligodendroglioma
with prominent neuronal differentiation [15], 7 anaplastic
oligodendrogliomas, 6 oligoastrocytomas, and 10 anaplastic oligoastrocytomas. All cases were reviewed and diagnosed according to the current WHO classification of thecentral nervous system [16]. Sections used for immunohistochemistry and genetic analyses were prepared fromformalin-fixed paraffin-embedded tissue specimens. The
study protocol was approved by the institutional ethics
committee and conducted according to the principles of the
Declaration of Helsinki.
Immunohistochemistry
Mutant IDH1R132H and wild type IDH1 protein expression
was determined immunohistochemically with paraffin-

123embedded tumor specimens. A streptavidin–biotin immunoperoxidase technique with anti-IDH1R132H antibody(mouse monoclonal, clone H09; Dianova GmbH, Hamburg, Germany) [8, 17] and anti-wild type IDH1 antibody(rat monoclonal, clone RcMab-1; Medical & Biological
Laboratories Co., Ltd., Nagoya, Japan) [18, 19] was carriedout. Histofine SAB-PO kit (Nichirei, Tokyo, Japan) and
VECTASTAIN Elite ABC Rat IgG Kit (Vector Laboratories, Burlingame, CA, USA) were used for H09 and
RcMab-1, respectively. Sections of 3-lm thickness wereautoclaved in 10 mM citrate buffer (pH 6.0) for 10 min at
121 "C for antigen retrieval. After blocking, the sections
were treated with optimally diluted primary antibodies(H09 1:50; RcMab-1 1:200) and then incubated overnightin a moist chamber at 4 "C. Immunoreactions were visualized with the staining kits mentioned above and 3,30 diaminobenzidine solution.
Direct DNA sequencing of IDH1 mutations
Genomic DNA was extracted from paraffin-embedded
sections, as described previously [20]. Briefly, regions ofinterest identified in a consecutive H&E-stained section
were manually scraped off from formalin-fixed, paraffinembedded tissue sections with a dry-heat sterilized spatula.
For case 9 and 20, DNA was extracted separately fromareas of astrocytoma and oligodendroglioma, and fromareas of oligodendroglioma and neuronal differentiation,
respectively. After deparaffinization with xylene and dehydration with ethanol, the tissue samples were digested
with proteinase K. The DNA was then amplified by PCR
using two primer sets for exon 4 of the IDH1 gene. The
sequences of the primers used to detect IDH1 mutations
have been reported previously [21]. PCR products werethen sequenced on a 3130xl Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA) with the Big Dye
Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) according to standard procedures.

Results
Clinical data, histological diagnosis, and the results ofimmunohistochemistry and genetic analyses are summarized in Table 1. There were 22 male and 21 female patients, ranging in age from 13 to 81 years (mean47.0 years, median 48 years). All the gliomas were locatedsupratentorially. Histology of diffuse astrocytomas(Fig. 1a), anaplastic astrocytomas (Fig. 1d), glioblastomas,
a glioblastoma with oligodendroglioma component(Fig. 1g), oligodendroglioma (Fig. 1j), anaplastic oligodendrogliomas, oligoastrocytomas, and anaplastic oligoastrocytomas (Fig. 2a, d, g, j) was conventional. In all theBrain Tumor Pathol
Table 1 Clinicopathological summary, immunohistochemical findings, and IDH1 status of diffusely infiltrating gliomas
Case

Histological diagnosis

Age

GenderLocationmIDH1R132H

RcMab-1

IDH1
mutation1

Diffuse astrocytoma

36

F

Right frontal lobe

??

??

G395A

2

Diffuse astrocytoma

30

F

Right temporal lobe

??

??

G395A

3

Diffuse astrocytoma

38

M

Left temporal lobe

-

?

-

4

Diffuse astrocytoma

16

M

Left frontal lobe

-

??

-

5

Anaplastic astrocytoma

61

M

Right temporal lobe

-

?

-

6

Anaplastic astrocytoma

56

M

Right frontal lobe

-

??

-

7
8

Anaplastic astrocytoma
Anaplastic astrocytoma

27
63

M
F

Left frontal lobe
Splenium of corpus
callosum

-

??
??

-

9

Glioneuronal tumor with neuropil-likeislands

49

F

Left parietal lobe

Astrocytoma: ?

Astrocytoma: ?/-G395A

Oligodendroglioma:??

Oligodendroglioma:?

10

Glioblastoma

65

F

Trigone of the leftlateral ventricle

-

??

-

11

Glioblastoma

71

M

Right temporal lobe

-

??

-

12

Glioblastoma

61

M

Right thalamus

-

??

-

13

Glioblastoma

55

M

Left frontal lobe

-

?

-

14

Glioblastoma

67

F

Left frontal lobe

??

??

G395A

15

Glioblastoma with oligodendroglioma
component

13

F

Right temporal lobe

??

??

G395A

16

Oligodendroglioma

58

F

?

?

G395A

17

Oligodendroglioma

22

F

Right temporal lobe
and insula
Right frontal lobe

??

?

G395A

18

Oligodendroglioma

40

F

Right frontal lobe

-

?

-

19

Oligodendroglioma

56

M

Right temporal lobe

?

?

G395A

20

Oligodendroglioma with prominent
neuronal differentiation

46

F

Right frontal lobe

Oligodendroglioma:??

Oligodendroglioma:
??

G395A

Neuronal element:?/-

Neuronal element:?/-

21

Anaplastic oligodendroglioma

62

M

Right frontal lobe

-

?

-

22

Anaplastic oligodendroglioma

81

F

Left frontal andparietal lobes

?

?

G395A

23

Anaplastic oligodendroglioma

46

F

Right frontal lobe

??

?

G395A

24

Anaplastic oligodendroglioma

74

F

Right temporal lobe

??

?

G395A

25

Anaplastic oligodendroglioma

54

F

Right cingulate
gyrus

-

??

-

26

Anaplastic oligodendroglioma

45

F

Right frontal lobe

??

??

G395A

27

Anaplastic oligodendroglioma

48

M

Left frontal lobe

??

??

G395A

28

Oligoastrocytoma

48

M

Right frontal lobe

??

??

G395A

29

Oligoastrocytoma

57

M

Left and rightfrontal lobes

??

??

G395A

30

Oligoastrocytoma

34

M

Left frontal lobe
and insula

??

??

G395A

31

Oligoastrocytoma

23

M

Left frontal lobe

??

??

G395A

32

Oligoastrocytoma

32

M

Right frontal lobe

??

??

G395A

33

Oligoastrocytoma

43

M

Right frontal lobe

??

??

G395A

34

Anaplastic oligoastrocytoma

16

F

Right frontal lobe

?

??

G395A

35

Anaplastic oligoastrocytoma

48

M

Left frontal andparietal lobes

?

?

G395A

36

Anaplastic oligoastrocytoma

34

M

Left and rightfrontal lobes

?/-

?/-

G395A

123

Brain Tumor Pathol
Table 1 continued
LocationmIDH1R132H

RcMab-1

IDH1
mutationM

Left temporal lobe

?

?

G395A

F

Left frontal lobe

??

??

G395A

55

F

Right frontal lobe

??

??

G395A

Anaplastic oligoastrocytoma

34

M

Left and rightfrontal lobes

??

??

G395A

41

Anaplastic oligoastrocytoma

67

F

Right insular gyrus

-

??

C394T

42

Anaplastic oligoastrocytoma

43

M

Left temporal lobe

-

??

-

43

Anaplastic oligoastrocytoma

39

F

Left frontal lobe

?

?

G395A

Case

Histological diagnosis

Age

Gender37

Anaplastic oligoastrocytoma

42

38

Anaplastic oligoastrocytoma

70

39

Anaplastic oligoastrocytoma

40??, strongly positive; ?, modestly positive; ?/-, weakly positive; -, negative

anaplastic oligoastrocytomas, both oligodendroglial andastrocytic tumor cells were intimately mixed. The GTNI
case comprises mostly glioma components with scantneuropil-like islands, and the former was anaplastic
oligoastrocytoma (Fig. 3a, d), in which the astrocytic andoligodendroglial components abutted with a clear border.
The case of oligodendroglioma with prominent neuronaldifferentiation was composed of a mixture of oligodendroglioma, gangliocytoma, and neurocytoma-like areas(Fig. 3g, j). The neurocytoma-like area consisted of roundtumor cells with a neuropil-like fibrillary matrix.
Diffuse astrocytomas, a glioblastoma, a glioblastoma
with oligodendroglioma component, oligodendrogliomas,
anaplastic oligodendrogliomas, and oligoastrocytomas, allof which had been demonstrated to have the IDH1 G395A
mutation, were immunohistochemically positive for bothmIDH1R132H and RcMab-1 (Fig. 1b, c, h, i). On the other
hand, all the diffuse gliomas other than anaplastic
oligoastrocytomas without IDH1 mutation weremIDH1R132H-negative and RcMab-1-positive (Fig. 1e, f).
The anaplastic oligoastrocytomas showed variable results. Eight cases had the IDH1 G395A mutation and threeof them (case 38, 39, 40) showed strong staining for bothmIDH1R132H and RcMab-1 (Fig. 2b, c). One case (case 34)
showed modest and strong positive staining for
mIDH1R132H and RcMab-1, respectively. Three cases (case35, 37, 43) showed modest staining for both mIDH1R132H
and RcMab-1 (Fig. 2e, f). One case (case 36) showedambiguous staining for both mIDH1R132H and RcMab-1(Fig. 2h, i). A case with the IDH1 C394T mutation (case41; Fig. 2k, l) and a case with no IDH1 mutation (case 42)
were negative for mIDH1R132H but positive for RcMab-1.
In the GTNI case, the oligodendroglioma-like area hadstrong and modest staining for mIDH1R132H and RcMab-1,
respectively (Fig. 3b, c). In contrast, the astrocytoma-likearea showed indistinct staining for mIDH1R132H and
RcMab-1 (Fig. 3e, f). On the other hand, the IDH1 G395A
mutation was detected in both the oligodendroglioma-like123

and astrocytoma-like areas. For the oligodendroglioma
case with prominent neuronal differentiation, the oligodendroglia-like tumor cells were positive for bothmIDH1R132H and RcMab-1 (Fig. 3h, i). In contrast, thestaining for mIDH1R132H and RcMab-1 in the neurocytoma-like component was ambiguous (Fig. 3k, l). However, the IDH1 G395A mutation was detected in both theoligodendroglioma and neurocytoma-like areas by DNA
sequencing.

Discussion
In this study we analyzed mutated and wild type IDH1
protein expression and the IDH1 gene status of 43 gliomas,
which encompassed almost all types of diffusely infiltrating gliomas listed in WHO classification. Anaplastic
oligoastrocytomas with the IDH1 G395A mutation had
variable staining intensity for mIDH1R132H among thecases. This finding disproved our previous belief that
gliomas with the IDH1 G395A mutation must be diffusely
and strongly positive for mIDH1R132H. Interestingly, there
was a tendency that cases that were strongly or weakly
positive for mIDH1R132H showed comparable immunoreaction to RcMab-1. This suggests that the expression ofboth mutant and wild type IDH1 is comprehensively
regulated and a glioma should not be regarded as IDH1mutation negative just because of ambiguous positive
staining of mIDH1R132H.
Although the anaplastic oligoastrocytomas were variably positive for mIDH1R132H, intra-tumor heterogeneity
was not detected in any of these cases. In contrast, the
GTNI and oligodendroglioma case with prominent neuronal differentiation showed differential staining intensity
for mIDH1R132H throughout the tumor components,
although the IDH1 G395A mutation was detected in every
component. The tumor components which were ambiguously or strongly positive for mIDH1R132H showed aBrain Tumor Pathol

Fig. 1 Representative histological features and immunohistochemical staining of diffuse astrocytoma (a–c case 2), anaplastic
astrocytoma (d–f case 6), glioblastoma with oligodendroglioma
component (g–i case 15), and oligodendroglioma (j–l case 16).
H&E stain (a, d, g, j), mIDH1R132H (b, e, h, k), and RcMab-1 (c, f, i,l). Diffuse astrocytoma, glioblastoma with oligodendroglioma component, and oligodendroglioma are positive for both mIDH1R132H and
RcMab-1. Anaplastic astrocytoma is negative for mIDH1R132H but
positive for RcMab-1

corresponding similar degree of immunoreaction to
RcMab-1. It is thought that IDH1 expression in each tumor
component is regulated under different mechanisms.
The inconsistency between the weak mIDH1R132H/
RcMab-1 staining and presence of the IDH1 G395A mutation in this study is difficult to explain. As tumor cells
showed weaker staining for RcMab-1 than that of nonneoplastic cells such as endothelia (Figs. 2i, 3f, l), thesuppression of whole IDH1 expression in the tumor cells
would be assumed. The reason why this inconsistency wasobserved only in anaplastic oligoastrocytomas, GTNI, andoligodendroglioma with prominent neuronal differentiation
was uncertain in this study.

To date, several monoclonal antibodies both specificand multi-specific for mutant and wild type IDH1/2 havebeen established [22]. Among them, the anti-wild type
IDH1 antibody RcMab-1 contributed greatly to thisstudy. Prior to this research, we compared three antiwild type IDH1 antibodies: clone W09 [17], RMab-3[23], and RcMab-1 [18, 19]. As a result, RcMab-1
showed the highest signal-to-background ratio (data notshown). Although direct sequencing keeps the best position in IDH mutation detection, we hereby emphasize
the utility of immunohistochemistry. A previous reportpresented glioma cases that were determined to be IDH1
wild type by DNA sequencing, nonetheless were123Brain Tumor Pathol

Fig. 2 Representative histological features and immunohistochemical staining of anaplastic oligoastrocytomas (a–c case 38, d–f case 43, g–i case 36, j–l case 41). H&E stain (a, d, g, j),
mIDH1R132H (b, e, h, k), and RcMab-1 (c, f, i, l). Staining of bothmIDH1R132H and RcMab-1 in cases 38, 43, and 36 are strongly,
modestly, and ambiguously positive, respectively. Case 41 is negative
for mIDH1R132H but positive for RcMab-1

immunopositive for IMab-1 an antibody against mutant
IDH1R132H. The authors in that paper explained that
DNA sequencing had not worked well with paraffinembedded materials because of contaminated samples or
low tumor cellularity [24]. Immunohistochemistry is amore readily available method and is useful even insmall and hypocellular samples.
Taking all the discussion into consideration, we propose
an evaluation procedure of IDH1 status by immunohistochemistry in diffusely infiltrating gliomas. If tumor cells
are strongly positive for mIDH1R132H, the tumor is judged

to have the IDH1 G395A mutation. If tumor cells arenegative for mIDH1R132H, the IDH1 G395A mutation is
judged to be absent. If tumor cells are weakly positive for
mIDH1R132H, immunohistochemistry for RcMab-1 shouldbe performed. If the tumor cells are strongly positive for
RcMab-1, the IDH1 G395A mutation is judged to be absent
on the grounds that IDH1 expression is not suppressed. A
false positive for the IDH1 G395A mutation due to nonspecific staining for mIDH1R132H should also be considered. If the tumor cells are weakly positive for bothmIDH1R132H and RcMab-1, DNA sequencing should be123Brain Tumor Pathol

Fig. 3 Representative histological features and immunohistochemical staining of glioneuronal tumor with neuropil-like islands(GTNI) (a–f case 9) and oligodendroglioma with prominent neuronaldifferentiation (g–l case 20). H&E stain (a, d, g, j), mIDH1R132H (b, e,h, k), and RcMab-1 (c, f, i, l). In the GTNI case, the oligodendroglioma-like area (a–c) is strongly and modestly positive for

mIDH1R132H and RcMab-1, respectively. In contrast, the astrocytoma-like area (d–f) shows indistinct staining for mIDH1R132H and
RcMab-1. In the oligodendroglioma with prominent neuronal differentiation case, the oligodendroglia-like area (g–i) is positive for bothmIDH1R132H and RcMab-1. In contrast, the staining for mIDH1R132H
and RcMab-1 in the neurocytoma-like component (j–l) is ambiguous

performed due to possible IDH1 suppression by unidentified regulatory mechanisms. This procedure will be usefulfor the practical evaluation of IDH1 status.

References

Acknowledgments We thank Mr. Koji Isoda and Mr. Yu Otaki(Gunma University) for their excellent technical assistance. This work
was supported in part by Platform for Drug Discovery, Informatics,
and Structural Life Science from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (Y.K.), and a Grant-in-Aid
for Young Scientists (B) (No. 24790346) from the Japan Society for
the Promotion of Science (to H.I.).
Conflict of interest

The authors have no conflict of interest.

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