Brain Tumor Pathol
DOI 10.1007/s10014-016-0260-x

ORIGINAL ARTICLE

Immunohistochemistry on IDH 1/2, ATRX, p53 and Ki-67
substitute molecular genetic testing and predict patient prognosis
in grade III adult diffuse gliomas
Shingo Takano1 • Eiichi Ishikawa1 • Noriaki Sakamoto1,2 • Masahide Matsuda1 •Hiroyoshi Akutsu1 • Masayuki Noguchi2 • Yukinari Kato3 • Tetsuya Yamamoto1 •Akira Matsumura1

Received: 21 February 2016 / Accepted: 2 March 2016Ó The Japan Society of Brain Tumor Pathology 2016

Abstract The molecular subgrouping of diffuse gliomas
was recently found to stratify patients into prognosticallydistinct groups better than histological classification.
Among several molecular parameters, the key molecules
for the subtype diagnosis of diffuse gliomas are IDH
mutation, 1p/19q co-deletion, and ATRX mutation; 1p/19q
co-deletion is undetectable by immunohistochemistry, but
is mutually exclusive with ATRX and p53 mutation in IDH
mutant gliomas. Therefore, we applied ATRX and p53
immunohistochemistry instead of 1p/19q co-deletion
analysis. The prognostic value of immunohistochemical
diagnosis for Grade III gliomas was subsequently investigated. Then, the same immunohistochmical diagnostic
approach was expanded for the evaluation of Grade II and
IV diffuse glioma prognosis. The results indicate
immunohistochemical analysis including IDH1/2, ATRX,
p53, and Ki-67 index is valuable for the classification ofdiffuse gliomas, which is useful for the evaluation of
prognosis, especially Grade III gliomas and lower-grade
gliomas (i.e., Grade II and III).
Keywords ATRX  Glioma  IDH 
Immunohistochemistry  Ki-67 index  p53& Shingo Takano
shingo4@md.tsukuba.ac.jp
1

Department of Neurosurgery, Faculty of Medicine,
University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-8575, Japan2

Department of Pathology, Faculty of Medicine, University ofTsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan3

Department of Regional Innovation, Tohoku University
Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku,
Sendai, Miyagi 980-8575, Japan

Introduction
Anaplastic astrocytomas, oligodendrogliomas, and mixed
oligoastrocytomas (i.e., Grade III gliomas) can be divided
into 3 prognostically different groups with respect to IDH
and ATRX mutations and 1p/19q status [1, 2]; this molecular subgrouping stratifies patients into prognosticallydistinct groups better than histological classification.
Consequently, the inclusion of molecular parameters in the
WHO definition of brain tumors is being planned and has
been forwarded as the ‘‘ISN-Haarlem’’ consensus [3].
Moreover, Caccellari et al. [4] classified diffuse glioma(Grade II–IV) into 7 different subtypes on the basis ofclinical and various molecular/genetic parameters using
TCGA data.
These molecular subtypes are clinically important,because treatment strategies can be planned with respect tomolecular subtype in conjunction with WHO grade. The
two major ways to identify these subtypes in a tumor
sample are direct interrogation of the mutated DNA itself
and immunohistochemical analysis to assess the effects ofthe mutated genes on proteins. Immunohistochemistry is alongstanding, affordable, robust, and widely available
technology. Therefore, immunohistochemical approacheswith defined protocols and materials have become an
essential means of assessing molecular genetic changes andthus, a critical component of current practice [5]. Among
several molecular parameters, the key molecules for thesubtype diagnosis of diffuse gliomas are IDH mutation, 1p/
19q co-deletion, and ATRX mutation. Among them, IDH
mutation [6] and ATRX mutation [7, 8] can be detected
accurately by immunohistochemistry. Immunohistochemistry shows that ATRX mutation results in loss of protein
expression, which is close to being mutually exclusive to
1p/19q co-deletion; moreover, all patients with 1p/19q co-123Brain Tumor Pathol

deletion carry a mutation in IDH1 or IDH2 [1]. In addition,
1p/19q co-deletion is close to being mutually exclusive to
p53 mutation [9, 10], which is also demonstrable byimmunohistochemistry [11]. Furthermore, 1p/19q co-deletion is close to being mutually exclusive with ATRX
mutation and p53 mutation with IDH mutation. Accordingly, the present study investigated the usefulness of the
key molecular parameters (IDH mutation and 1p/19q codeletion for IDH, ATRX, and p53) for the immunohistochemical diagnosis of diffuse gliomas.

Materials and methods
Patients
Fifty-three patients who underwent primary surgery
between 1994 and 2011 at Tsukuba University Hospitalwere included. The mean patient age at the time of primary
surgery was 48.7 ± 14.5 years (range 20–82 years). All
tumors were diagnosed as Grade III gliomas according to
the WHO classification, including 26 anaplastic astrocytomas, 8 anaplastic oligodendrogliomas, and 19 anaplastic
oligoastrocytomas. Postoperative therapies were uniform.
All patients were treated with maximal surgical resection,
followed by 54–60 Gy radiation and ACNU-based
chemotherapy. Median follow-up was 53.7 months (range
6–179 months). Informed consent was obtained from eachpatient or their guardian to obtain samples and use theassociated data for analysis.
To extend our investigation, Grade II and IV samples
were collected. All patients underwent primary surgery at
Tsukuba University Hospital (Grade II between 1994 and2004 and Grade IV between 2008 and 2010). There were
48 Grade IV samples including 37 primary glioblastomas
and 11 secondary glioblastomas as well as 35 Grade II
samples including 25 diffuse astrocytomas, 5 oligoastrocytomas, and 5 oligodendrogliomas. Postoperative therapies were uniform. For Grade IV tumors, the patients
received 60 Gy radiation therapy, followed by ACNU andtemozolomide-based chemotherapy. Patients with Grade IItumors underwent no further treatment after surgery,except for resection and radiation therapy in cases ofrecurrence.
Sample preparation
Tumor samples were removed intraoperatively, and themost viable part of an area devoid of macroscopicallyevident necrosis was taken as a specimen. The specimen
was divided into 2: one part was fixed in 10 % formalin,
and the other was frozen for subsequent analysis.

123

Immunohistochemistry
IDH mutation was determined by double immunohistochemistry using 2 mutation-specific anti-IDH antibodies(i.e., HMab-1 and MsMab-1) in gliomas in paraffin-embedded tumor specimens as described previously [6].
Briefly, IDH1-R132H protein expression was determinedusing HMab-1 at 5 lg/mL and an LSAB2 kit (Dako,
Glostrup, Denmark), and the expressions of the othermutant proteins in paraffin sections were determined using
MsMab-1 at 5 lg/mL and an Envision ? kit (Dako).
MsMab-1 recognizes several IDH mutations, namely
IDH1-R132S, IDH1-R132G, IDH2-R172S, IDH2-R172G,
and IDH2-R172M, as well as IDH1-R132H [12]. The
expression of IDH mutations was determined by semiquantitatively by assessing the proportions of positively
stained tumor cells. Cases in which C10 % cells were
stained were defined as positive.
All sections were immunostained with Ki-67 antibody(Immunotech Laboratories Inc., Monrovia, CA, USA), p53
antibody (clone DO7, Dako), and CD34 antibody (Dako) as
described previously [13]. The positivity of nuclei for Ki67 and p53 was determined by counting at least 1000 tumor
cells in a homogeneously stained area. For both Ki-67 andp53, cases with C10 and \10 % stained cells were defined
as positive and negative, respectively. The immunohistochemical cut-off of p53 mutation was defined as strong p53
immunoreactivity in [10 % of cells, which provides themost accurate prediction of mutation [11].
ATRX staining
All sections were immunostained with a commerciallyavailable polyclonal antibody (HPA001906, SigmaAldrich, St. Louis, MO, USA) at 0.5 lg/mL and an LSAB2
kit (DAKO) with Tris–EDTA antigen retrieval buffer (pH9.0). Preliminary staining with Tris–EDTA buffer (pH 9.0)
is superior to citrate buffer (pH 6.0) as an antigen retrieval
method. Physiologically, ATRX protein is ubiquitously
expressed in cell nuclei. Mutations in its gene result in aloss of nuclear protein expression in tumor cells but
retained expression in non-tumor cells (e.g., endothelial
and pre-existing glial cells), serving as a positive internal
control [14]. Cases with C50 % stained cells (???) were
defined as ATRX loss-negative, and cases with \50 %stained cells (??; C10, ?; \10 %, -; 0 %) accompaniedwith endothelial cell-positive staining were defined as
ATRX loss.
Statistical analysis
Overall survival was calculated from the time of surgery
until death or the last follow-up and was compared among

Brain Tumor Pathol

groups using the Kaplan–Meier method and the log-rank
test. The Cox proportional hazards model was used to test
prognostic factors in univariate and multivariate analyses.
The results are expressed as relative risk with 95 % confidence intervals (CIs).

Results
ATRX staining
A total of 137 gliomas including 35, 53, and 48 Grade II–
IV, respectively, were stained Fig. 1). ATRX loss was
frequently observed in secondary glioblastoma (53.6 %),anaplastic astrocytoma (80.8 %), anaplastic oligoastrocytoma (68.4 %), and diffuse astrocytoma (56.0 %) and lessfrequently in primary glioblastoma (26.3 %), anaplastic
oligodendroglioma (37.5 %), oligoastrocytoma (20.0 %),and oligodendroglioma (0 %) (Table 1).
Molecular classification by immunohistochemistry
Recent investigations support IDH1/2 mutation, p53
mutation, and ATRX loss separately for the classificationof biologically distinct groups of gliomas, and indicate
improved outcomes using this classification [1, 2, 4, 8].
Therefore, Kaplan–Meier analysis was used to evaluate theoutcomes of 53 patients with Grade III gliomas following
stratification according to IDH1/2 mutation status (Fig. 2a).
IDH mutation (n = 28) was associated with longer survival
relative to wild-type IDH (IDH1/2 wild-type, n = 25)[median overall survival (OS) = 60.2 vs. 33.9 months;
log-rank test, P \ 0.01, hazard ratio (HR): 2.3823 (95 %Grade IV

CI 1.2711–5.2452)]. In addition, p53 mutation and ATRX
loss status were evaluated together, and OS was comparedamong IDH/p53/ATRX mutants (n = 11), IDH mutant/others (n = 17) and IDH wild-type (n = 25); their median
OS was 43.3, 94.0, and 33.9 months, respectively. The IDH
mutant/other group had significantly longer survival thanthe other groups (log-rank test P \ 0.001) (Fig. 2b).
There was still overlap in OS between the IDH/p53/
ATRX mutant group and the IDH wild-type group. We
subsequently investigated the prognostic factors in IDH
wild-type Grade III gliomas. Among various genetic,
clinical, and biological factors, multivariate analysis
demonstrated Ki-67 positivity (C10 %) was a significant
factor[P = 0.0111,
HR:
4.0686(95 %CI
1.3776–12.0161)] (Table 2). Furthermore, we divided IDH
wild-type into 2 groups: the IDH wild-type/high Ki-67 and
IDH wild-type/low Ki-67 groups.
Prognostic analysis of Grade III gliomas using
the new molecular classification
by immunohistochemistry
The gliomas were classified into 4 final groups: Group A,
IDH mutant/others (n = 17); Group B, IDH wild-type/Ki67 low (n = 11); Group C, IDH/p53/ATRX mutant(n = 11); and Group D, IDH wild-type/Ki-67 high(n = 14). The Kaplan–Meier curves clearly showed prognosis differed among the 4 groups: Group A had the best
prognosis (median OS: 117.0 months), followed by GroupB (99.5 months), Group C (65.2 months), and Group D(28.7 months) (P \ 0.001, Fig. 3a). Progression-free survival showed a similar trend: Group A had the best median

Grade III

Grade II

p-GBM

AO

Oligo

s-GBM

AA

DA

+++

-

Fig. 1 ATRX staining with diffuse gliomas. ??? positive, negative, p-GBM primary glioblastoma, AO anaplastic oligodendroglioma, oligo oligodendroglioma, s-GBM secondary glioblastoma,

AA anaplastic astrocytoma, DA diffuse astrocytoma. Please note
endothelial cells were positive in all sections

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Brain Tumor Pathol
Table 1 ATRX staining pattern in Grade II, III and IV glioma

Table 2 Prognostic factors for IDH wild type Grade III glioma

Number

???

??

?, -

Mut %

Primary

38

28

9

1

26.3

Secondary

11

4

5

2

53.6

AA

26

5

14

7

80.8

AOA

19

6

7

6

68.4

Mut: 1

AO

8

5

2

1

37.5

WT: 0

DA

25

11

10

4

56.0

OA

5

4

1

0

20.0

\55: 0

Oligo

5

5

0

0

0

Sex
Male: 1GBM

P valueHR

95 % CI

0.75950.84180.2795–2.5356

0.28210.58430.2195–1.5555

0.19851.80580.7336–4.4455

0.82140.90150.3663–2.2184

0.2577

3.21800.4253–24.3511

0.4726

0.71810.2908–1.7729

0.57901.29640.5181–3.2439

0.00574.4656

1.546–12.902

0.04912.56381.0038–6.5484

0.2271

1.73260.7101–4.2171

0.0111

4.06861.3776–12.0161

0.11172.18260.8341–5.7108

Univariate
p53[10 %: 1\10 %: 0

Grade III

Grade II

ATRX

Age
[55: 1

Female: 0
RemovalSurvival %

A

100
90
80
70
60
50
40
30
20
10
0

Non-total: 1
Total: 0

Mutant type (n28)Location
Others: 1
Frontal: 0
MGMT

Wild type (n25)[10 %: 1

p<0.01
50

0

100

150

200

Time (month)

Survival %B\10 %: 0
Ki-67[10 %: 1

100
90
80
70
60
50
40
30
20
10
0

\10 %: 0
Density[30: 1\30: 0VEGF?: 1
p<0.001
0

50

100

150

200

Time (month)Fig. 2 a Kaplan–Meier curves of overall survival in 53 patients with
Grade III glioma following stratification by IDH1/2 mutation status.
b Kaplan–Meier curves following stratification by IDH1/2, p53, andATRX mutation statusprogression-free survival (67.2 months), whereas Group D
had the worst (12.2 months) (P \ 0.001, Fig. 3b; Table 3).
Kaplan–Meier curves based on classical morphologicaldiagnosis, which included anaplastic oligodendroglioma(n = 9), anaplastic oligoastrocytoma (n = 18), andanaplastic astrocytoma (n = 26), demonstrated significant
differences in prognosis; however, differences in survival
were unclear compared to the curve based on the proposed
molecular diagnosis by immunohistochemistry, especially

123

-: 0
Multivariate
Ki-67[10 %: 1\10 %: 0
Density[30: 1\30: 0

between anaplastic oligoastrocytoma and anaplastic astrocytoma, (P \ 0.001, Fig. 3c).
The initial morphological diagnosis and immunohistochemical molecular diagnosis were compared (Fig. 4a).
Most anaplastic oligodendrogliomas were classified into
Group A, and anaplastic oligoastrocytomas and anaplastic
astrocytomas were classified equally into the 4 groups.

Brain Tumor Pathol

Among the groups, Group D had the poorest prognosis,
close to that of glioblastomas; the profile of Group D was
further investigated. First, a central review of all samples
was performed by another neuropathologist using othersections of the sample. Second, other molecular parameters
related to tumor biology were evaluated immunohistochemically, including VEGF, HIF-1a, podoplanin, phosphorylated Akt staining [15, 16], and microvascularvascular density stained by CD34. Among Group D(n = 14, Table 4), in addition to IDH mutation, the following other mutations were observed: p53 mutation in 3
cases and ATRX loss in 7 cases. The central review
demonstrated necrosis and microvascular proliferation in 2
and 4 cases, respectively, (Table 4; cases 1–4), werediagnosed as 2 glioblastomas and 2 glioblastoma with anoligodendroglioma component, respectively. Among these
4 cases, 3 were ATRX wild-type and 1 was 1p/19q codeletion–negative according to FISH analysis and othermolecular profile expression (i.e., VEGF, HIF-1a, podoplanin, and phosphorylated Akt positivity), suggesting thepossibility of Grade IV at the initial diagnosis.

Survival %

A 100
90
80
70
60
50
40
30
20
10
0

IDH mut / others (n17)

IDH WT / Ki-67>10% (n 14)

IDH WT / Ki-67<10% (n11)
IDH mut / p53 mut / ATRX mut (n11)OS

p<0.001

0

50

100

150

200

Time (month)

Survival %

B 100
90
80
70
60
50
40
30
20
10
0

IDH mut / others (n17)

IDH WT / Ki-67<10% (n11)

IDH WT / Ki-67>10% (n 14)

IDH mut / p53 mut / ATRX mut (n11)

PFS
0

20

p<0.001
40

60

80

100

120

140

160

180

Time (month)

Survival %

C 100
90
80
70
60
50
40
30
20
10
0

Anaplastic oligodendroglioma (n9)p<0.001
Anaplastic oligoastrocytoma (n18)OS0Anaplstic astrocytoma (n26)
50

100

150

200

Time (month)Fig. 3 a Kaplan–Meier curves of overall survival in 53 patients with
Grade III gliomas following stratification by IDH1/2, p53, ATRX
mutation status, and Ki-67 positivity. b Kaplan–Meier curves ofprogression-free survival. c Kaplan–Meier curves of overall survival
according to the three classic histological groups
Table 3 Detail molecule
profile in molecular diagnosis
group

Prognostic analysis of Grade II and IV gliomas using
the molecular classification
by immunohistochemistry
The same molecular classification by immunohistochemistry was adapted for Grade II and IV gliomas, and its
effectiveness for prognosis analysis was evaluated. There
were 20, 9, 6, and 0 Grade II gliomas classified into Groups
A–D, respectively, (n = 35, Fig. 4b). All histologicaloligodendrogliomas were in Group A. Oligoastrocytomas
were divided equally into Groups A and B. Astrocytomas
were divided equally among Groups A–C. The prognosis
of Group C tended to be shorter than that of other groups(Fig. 5a, b).

Ki67 [10 %Med OS (mo)

8

9

117.0

10

0

99.5

11

5

65.2

7

14

28.7

20

36

28

4

14

11

84.0

20

4

13

0

104.9

17

17

17

5

52.0

14

14

3

7

14

25.3

88

88

28

51

30

n

IDH mut

P53 mut

Group A17

17

3Group B

11

0

3

Group C

11

11

11

Group D

14

0

3

Total

53

28

Group A

37

37

Group B

20

Group C

17

Group D
Total

ATRX loss

Grade III

Grade II ? III

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Brain Tumor Pathol

A

B

Initial diagnosis
AO

AOA

AA

8

19

26

11

17

11

Group A Group B Group C
AA
AA
AO-IDH WT -IDH mut

C

14
GBM

Initial diagnosis

20

D

25

9

0

6

Secondary GBM
11

37

0

A

5

Initial diagnosis

Primary GBM

4OA

Group A Group B Group C Group D
A
A
GBM
O
-IDH WT -IDH mut

Initial diagnosis

2

O
5

4

31

Group A Group B Group C Group D
AA
AA
GBM
AO
-IDH WT-IDH mut

32

were classified into Group D; 2 and 4 were classified
into Groups A and B, respectively, (Fig. 4c). Becauseof the few cases in Group D, the molecular classification by immunohistochemistry did not demonstratesignificant superiority for determining the prognosis ofprimary glioblastoma (Fig. 5c). In contrast, secondary
glioblastomas were distributed equally among groups:
4, 3, 2, and 2 in Groups A–D, respectively, (Fig. 4d);
there was a significant difference in the prognosis
among groups (Fig. 5d).
As it was recently reported that there is no genetic difference between Grade II and III gliomas [17–19], we
evaluated our classification by combining Grade II and III
gliomas as lower-grade gliomas. Among the 88 lowergrade gliomas, 37, 20, 17, and 14 were classified into
Groups A–D, respectively. Median OS in Groups A–D was
84.0, 104.9, 52.0, and 25.3 months, respectively; OS wassignificantly longer in Groups A and B than Groups C and
D (Fig. 6; Table 3) despite different initial treatments
between Grade II and III gliomas.

2Group A Group B Group C Group D
AA
AA
AO
GBM
-IDH WT-IDH mut

Fig. 4 Comparison of initial morphological diagnosis and immunohistochemical molecular diagnosis. a Grade III gliomas; b Grade II
gliomas; c Grade IV primary glioblastomas; d Grade IV secondary
glioblastomas; AO anaplastic oligodendroglioma, AOA anaplastic
oligoastrocytoma, AA anaplastic astrocytoma, O oligodendroglioma,
OA oligoastrocytoma, A astrocytoma, GBM glioblastoma

Grade IV gliomas (n = 48) included 37 and 11
primary and secondary glioblastomas, respectively.
The majority of primary glioblastomas (31/37 cases)

Discussion
The results of the present study indicate that combined
immunohistochemistry including IDH1/2, ATRX, p53, andKi-67 index is useful for the molecular classification ofdiffuse gliomas, which is useful for the evaluation of
prognosis, especially Grade III and lower-grade gliomas(i.e., Grade II ? III). Although our immunohistochemical
approach does not replace the genetic testing completely, it
is a practical and a powerful means of assessing molecular
genetic changes.

Table 4 Molecular profile of Group D
IDH

Status

Ki-67 %

P53 mut

ATRX loss

Pathol 1

Pathol 2

NecrosisMVP

Density

VEGF

HIF1

Podoplanin

PAkt

1

WT

17.1

?

-

AOA

GBMO

?

?

31

-

?

?

?

2

WT

30.8

-

-

AOA

GBMO

?

?

107

?

?

?

-

3

WT

20.5

-

-

AO

GBM

?

-

81

-

?

?

-

4

WT

23.8

-

?

AA

GBM

?

-

32

?

?

?

?

5

WT

29

?

?

AOA

GBMO

?

-

45

-

?

?

?

6

WT

10.6

-

?

AOA

AOA

-

-

11

-

-

-

?

7

WT

36.4

-

?

AA

AA

-

-

96

?

?

?

?

8

WT

14.2

-

?

AOA

AOA

-

-

24

?

?

?

?

9

WT

10.6

-

?

AOA

AOA

-

-

65

-

?

?

?

10

WT

20.1

-

?

AA

AA

-

-

18

-

?

-

?

11
12

WT
WT

12.1
12.2

?
-

-

AA
AA

AA
AA

-

-

15
8

?
?

?
-

?
-

?
-

13

WT

26.1

-

-

AO

AO

-

-

32

-

?

?

-

14

WT

52.1

-

-

AOA

AOA

-

-

28

?

?

-

-

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Brain Tumor Pathol

B

100
90
80
70
60
50
40
30
20
10
0

Oligodendroglioma (n5)Oligoastrocytoma (n5)Survival %Survival %A

Astrocytoma (n25)
0

50

100

150

200

100
90
80
70
60
50
40
30
20
10
0

Grade IINS
IDH mut / others (n20)

IDH WT / Ki-67<10% (n9)IDH mut / p53 mut / ATRX mut (n6)

0

250

50

150

200

250

D

100
90
80
70
60
50
40
30
20
10
0

Primary GBM

NS
IDH mut / others (n2)Survival %Survival %C100Time (month)

Time (month)

IDH WT / Ki-67<10% (n4)IDH WT / Ki-67>10% (n31)

0

20

40

60

80

100

120

Sec GBM

100
90
80
70
60
50
40
30
20
10
0

p<0.01
IDH mut / p53 mut/ ATRX mut (n2)
IDH mut / others (n4)IDH WT / Ki-67<10% (n3)IDH WT / Ki-67>10% (n2)

0

20

40

60

80

100

Time (month)

Time (month)

Survival (%)

Fig. 5 Kaplan–Meier curves of overall survival. a Grade III gliomas with initial morphological diagnosis; b Grade II gliomas with moleculardiagnosis; c Grade IV primary glioblastomas with molecular diagnosis; d Grade IV secondary glioblastomas with molecular diagnosis

100
90
80
70
60
50
40
30
20
10
0

IDH mut / others (n37)

IDH WT / Ki-67<10% (n20)
IDH mut / p53 mut / ATRX mut (n17)
IDH WT / Ki-67>10% (n 14)

0

50

100

150

200

250

Time (month)Fig. 6 Kaplan–Meier curves of overall survival in 88 lower-grade(i.e., Grade II ? Grade III) gliomas following stratification according
to IDH1/2, p53, ATRX mutation status, and Ki-67 positivity

Immunohistochemistry-based molecular diagnosis
The molecular diagnosis for diffuse gliomas has definitive
treatment and prognostic importance [1, 2, 4, 8]. The
original molecular markers were IDH mutation status and1p/19q LOH status, following by ATRX status [1].
The present study revealed the frequency of immunohistochemical ATRX loss (i.e., ATRX mutation); it occurred
frequently in Grade IV secondary glioblastoma; in the
majority of Grade III anaplastic astrocytomas (80.8 %), Grade
III anaplastic oligoastrocytomas (68.4 %), and Grade II

astrocytomas (56 %); infrequently in primary glioblastomas(26.3 %), Grade III anaplastic oligodendrogliomas (37.5 %),and Grade II oligodendrogliomas (20 %); and in 0 % ofoligoastrocytomas. These results are similar to those of previous studies [7, 8] reporting ATRX loss in 45–73 % of Grade
III anaplastic astrocytomas, 4 % of primary glioblastomas,
27–68 % of Grade III anaplastic oligodendrogliomas, and
14 % of Grade II oligodendrogliomas.
With regard to the immunohistochemical cut-off for p53
mutation, strong p53 immunoreactivity in C10 % of cells isreported to provide the most accurate prediction of mutation[11]. Using this cut-off, it was found that 52 of 55
immunopositive cases harbored a mutation, whereas only 14
of 102 immunonegative cases showed mutations; the sensitivity and specificity of this cut-off were 78.8 and 96.7 %,respectively. Therefore, p53 immunohistochemistry was
proposed as a moderately sensitive and highly specificmarker for the prediction of p53 mutation. Because p53
mutation is almost invariably associated with IDH1/2
mutations in Grade II astrocytomas and Grade III anaplastic
astrocytomas [20, 21] and mutually exclusive 1p/19q codeletion [9, 10], we selected p53 as a molecular marker.
Routine immunohistochemistry can be used to evaluate
ATRX loss, p53 protein accumulation, and IDH1R132H
mutation but not 1p/19q co-deletion; this may enable theclassification of diffuse glioma outcomes. There is strong
evidence of relationships among ATRX loss, mutually123Brain Tumor Pathol

exclusive 1p/19q co-deletion [8, 22], and p53 mutually
exclusive 1p/19q co-deletion [9, 10], suggesting the usefulness of ATRX and p53 immunohistochemistry without
1p/19q co-deletion analysis. In addition, we focused on Ki67, which is a classic and reliable indicator of cancer cellproliferation activity used in routine clinical investigation;
it predicts poorer prognosis in patients with gliomas [23,24]. It was recently reported that high Ki-67 expression isdominant in IDH1/2 wild-type gliomas [25] and that low
Ki-67 expression is associated with IDH1 mutations inprimary glioblastomas [26]. Ki-67 expression in combination with IDH1/2 mutation status quickly indicates prognosis in gliomas [27]. Olar et al. [28] report that the mitotic
index is significantly associated with outcome in IDH wildtype tumors but not in IDH mutant tumors. These findings
demonstrate the relationship between Ki-67 expression and
IDH1/2 mutations in gliomas. On the basis of these results,
Ki-67 expression was applied as a molecular marker of
IDH wild-type gliomas. Application of Ki-67 labelingindex to separate IDH-wild type gliomas is important,because IDH-wild-type gliomas are composed of heterogeneous group [4, 20].
Molecular classification by immunohistochemistry
predicts glioma prognosis
Finally, we selected IDH, p53, ATRX, and Ki-67 as
immunohistochemical molecular markers. The 53 histological Grade III cases were divided into 4 groups. First,
Group C, characterized by IDH positivity (mutant type),
p53 positivity (mutant type), and ATRX loss (mutant type),suggesting molecular astrocytomas, exhibited intermediate
prognosis. Group A, characterized by IDH positivity(mutant type) other than Group C, suggesting molecularoligodendroglioma, exhibited the best prognosis. Third,
Group B, characterized by IDH negativity (wild type) andKi-67 \10 % demonstrated intermediate prognosis; 10 of
11 cases in this group demonstrated ATRX loss (mutanttype), suggesting molecular astrocytomas. Finally Group
D, which was characterized by IDH negativity (wild type)
and Ki-67 C10 %, suggesting molecular glioblastoma,demonstrated the poorest prognosis.
A similar molecular classification by immunohistochemistry was applied for Grade II and IV gliomas. There
were no Grade II gliomas in Group D. Group C tended tohave poorer prognosis than the other groups. Compared toclassical histological diagnosis, tumors with molecularoligodendroglioma (Group A) were predominant. In addition, this molecular classification could not predict prognostic group for primary glioblastomas. Regarding theprediction of the prognosis of secondary glioblastoma, whichmainly depends on IDH status, Groups A and C (IDH

123

mutant) tended to have good prognosis, whereas Groups B
and D (IDH wild-type) tended to have poorer prognosis.
However, molecular classification by immunohistochemistry for lower-grade gliomas again demonstrated theusefulness of the present classification for the prediction of
prognosis. Group A tumors suggesting molecular oligodendroglioma exhibited best prognosis, Group C tumors suggesting molecular astrocytoma exhibited intermediate
prognosis, and Group D tumors suggesting molecularglioblastoma exhibited the poorest prognosis despite different initial treatments (e.g., radiation and chemotherapy).
These results suggest our molecular classification based on
immunohistochemical analysis is useful for predicting caseswith good prognosis among lower-grade gliomas. Accordingly, lower-grade gliomas with poor prognosis according to
the molecular classification should receive more intensive
treatment. The group of lower-grade gliomas with the best
prognosis had a median OS of 117 months with current
treatment strategies. Meanwhile, the median OS of the groupwith intermediate prognosis in Group C was only 55 months;
this group possessed 3 molecular mutations of IDH, p53, andATRX, suggesting a new molecular target for the tumor [29–32]. Group D exhibited the poorest prognosis, with a median
OS of only 25.3 months and should, therefore, be treated as
glioblastoma.

Conclusion
The molecular immunohistochemical classification
including IDH, p53, ATRX, and Ki-67 showed that thegroup of molecular oligodendrogliomas had the best
prognosis, followed by the molecular group of astrocytomas; meanwhile, the molecular glioblastoma group had
the poorest outcome among Grade III and lower-grade
gliomas.
Acknowledgments We thank Yoshiko Tsukada and Makiko
Miyakawa for their excellent technical assistance. This work was
supported in part by Grants-in-Aid for Scientific Research (to S. T.,
No. 15H04947) from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan; and by the Japan Brain Foundation(to S. T.) and the Japanese Foundation for Multidisciplinary Treatment of Cancer (to S. T.).
Compliance with ethical standards
Conflict of interest

The authors declare no conflicts of interest.

References
1. Reuss DE, Sahm F, Schrimpf D, Wiestler B, Capper D, Koelsche
C, Schweizer L, Korshunov A, Jones DT, Hovestadt V, Mittelbronn M, Schittenhelm J, Herold-Mende C, Unterberg A, Platten
M, Weller M, Wick W, Pfister SM, von Deimling A (2015)

Brain Tumor Pathol
ATRX and IDH1-R132H immunohistochemistry with subsequent
copy number analysis and IDH sequencing as a basis for an‘‘integrated’’ diagnostic approach for adult astrocytoma, oligodendroglioma and glioblastoma. Acta Neuropathol 129:133–146
2. Weller M, Weber RG, Willscher E, Riehmer V, Hentschel B,
Kreuz M, Felsberg J, Beyer U, Löffler-Wirth H, Kaulich K,
Steinbach JP, Hartmann C, Gramatzki D, Schramm J, Westphal
M, Schackert G, Simon M, Martens T, Boström J, Hagel C, Sabel
M, Krex D, Tonn JC, Wick W, Noell S, Schlegel U, Radlwimmer
B, Pietsch T, Loeffler M, von Deimling A, Binder H, Reifenberger G (2015) Molecular classification of diffuse cerebral
WHO grade II/III gliomas using genome- and transcriptome-wide
profiling improves stratification of prognostically distinct patientgroups. Acta Neuropathol 129:679–693
3. Louis DN, Perry A, Burger P, Ellison DW, Reifenberger G, vonDeimling A, Aldape K, Brat D, Collins VP, Eberhart C, FigarellaBranger D, Fuller GN, Giangaspero F, Giannini C, Hawkins C,
Kleihues P, Korshunov A, Kros JM, Beatriz Lopes M, Ng HK,
Ohgaki H, Paulus W, Pietsch T, Rosenblum M, Rushing E,
Soylemezoglu F, Wiestler O, Wesseling P (2014) International
Society Of Neuropathology-Haarlem. International Society Of
Neuropathology-Haarlem consensus guidelines for nervous system tumor classification and grading. Brain Pathol 24:429–435
4. Ceccarelli M, Barthel FP, Malta TM et al (2016) Molecularprofiling reveals biologically discrete subsets and pathways ofprogression in diffuse glioma. Cell 164:550–563
5. Tanboon J, Williams EA, Louis DN (2015) The Diagnostic use of
immunohistochemical surrogates for signature molecular genetic
alterations in gliomas. J Neuropathol Exp Neurol 75:4–18.
doi:10.1093/jnen/nlv009
6. Takano S, Kato Y, Yamamoto T, Liu X, Ishikawa E, Kaneko
MK, Ogasawara S, Matsuda M, Noguchi M, Matsumura A (2015)
Diagnostic advantage of double immunohistochemistry using two
mutation-specific anti-IDH antibodies (HMab-1 and MsMab-1) ingliomas. Brain Tumor Pathol 32:169–175
7. Jiao Y, Killela PJ, Reitman ZJ, Rasheed AB, Heaphy CM, deWilde RF, Rodriguez FJ, Rosemberg S, Oba-Shinjo SM, Nagahashi Marie SK, Bettegowda C, Agrawal N, Lipp E, Pirozzi C,
Lopez G, He Y, Friedman H, Friedman AH, Riggins GJ, Holdhoff M, Burger P, McLendon R, Bigner DD, Vogelstein B,
Meeker AK, Kinzler KW, Papadopoulos N, Diaz LA, Yan H(2012) Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine
the classification of malignant gliomas. Oncotarget 3:709–722
8. Wiestler B, Capper D, Holland-Letz T, Korshunov A, vonDeimling A, Pfister SM, Platten M, Weller M, Wick W (2013)
ATRX loss refines the classification of anaplastic gliomas andidentifies a subgroup of IDH mutant astrocytic tumors with better
prognosis. Acta Neuropathol 126:443–451
9. Ohgaki H, Kleihues P (2005) Population-based studies on
incidence, survival rates, and genetic alterations in astrocytic
and oligodendroglial gliomas. J Neuropathol Exp Neurol
64:479–489
10. Thon N, Eigenbrod S, Grasbon-Frodl EM, Ruiter M, Mehrkens
JH, Kreth S, Tonn JC, Kretzschmar HA, Kreth FW (2009) Novel
molecular stereotactic biopsy procedures reveal intratumoral
homogeneity of loss of heterozygosity of 1p/19q and TP53
mutations in World Health Organization grade II gliomas.
J Neuropathol Exp Neurol 68:1219–1228
11. Takami H, Yoshida A, Fukushima S, Arita H, Matsushita Y,
Nakamura T, Ohno M, Miyakita Y, Shibui S, Narita Y, Ichimura
K (2015) Revisiting TP53 Mutations and Immunohistochemistry—a comparative study in 157 diffuse gliomas. Brain Pathol
25:256–265
12. Kaneko MK, Ogasawara S, Kato Y (2013) Establishment of amulti-specific monoclonal antibody MsMab-1 recognized both
IDH1 and IDH2 mutations. Tohoku J Exp Med 230:103–109

13. Takano S, Kato Y, Yamamoto T, Kaneko MK, Ishikawa E,
Tsujimoto Y, Matsuda M, Nakai K, Yanagiya R, Morita S,
Tsuboi K, Matsumura A (2012) Immunohistochemical detectionof IDH1 mutation, p53, and internexin as prognostic factors of
glial tumors. J Neurooncol 108:361–373
14. Haberler C, Wöhrer A (2014) Clinical Neuropathology practicenews 2-2014: ATRX, a new candidate biomarker in gliomas. Clin
Neuropathol 33(2):108–111
15. Birner P, Pusch S, Christov C, Mihaylova S, ToumangelovaUzeir K, Natchev S, Schoppmann SF, Tchorbanov A, Streubel B,
Tuettenberg J, Guentchev M (2014) Mutant IDH1 inhibits PI3K/
Akt signaling in human glioma. Cancer 120:2440–2447
16. Mashiko R, Takano S, Ishikawa E, Yamamoto T, Nakai K,
Matsumura A (2011) Hypoxia-inducible factor 1a expression is aprognostic biomarker in patients with astrocytic tumors associated with necrosis on MR image. J Neurooncol 102:43–50
17. Cancer Genome Atlas Research Network, Brat DJ et al (2015)
Comprehensive, integrative genomic analysis of diffuse lowergrade gliomas. N Engl J Med 372:2481–2498
18. Eckel-Passow JE, Lachance DH, Molinaro AM, Walsh KM,
Decker PA, Sicotte H, Pekmezci M, Rice T, Kosel ML, Smirnov
IV, Sarkar G, Caron AA, Kollmeyer TM, Praska CE, Chada AR,
Halder C, Hansen HM, McCoy LS, Bracci PM, Marshall R,
Zheng S, Reis GF, Pico AR, O’Neill BP, Buckner JC, Giannini C,
Huse JT, Perry A, Tihan T, Berger MS, Chang SM, Prados MD,
Wiemels J, Wiencke JK, Wrensch MR, Jenkins RB (2015)
Glioma groups based on 1p/19q, IDH, and TERT promoter
mutations in tumors. N Engl J Med 372:2499–2508
19. Suzuki H, Aoki K, Chiba K, Sato Y, Shiozawa Y, Shiraishi Y,
Shimamura T, Niida A, Motomura K, Ohka F, Yamamoto T,
Tanahashi K, Ranjit M, Wakabayashi T, Yoshizato T, Kataoka K,
Yoshida K, Nagata Y, Sato-Otsubo A, Tanaka H, Sanada M,
Kondo Y, Nakamura H, Mizoguchi M, Abe T, Muragaki Y,
Watanabe R, Ito I, Miyano S, Natsume A, Ogawa S (2015)
Mutational landscape and clonal architecture in grade II and III
gliomas. Nat Genet 47:458–468
20. Ichimura K, Narita Y, Hawkins CE (2015) Diffusely infiltrating
astrocytomas: pathology, molecular mechanisms and markers.
Acta Neuropathol 129:789–808
21. Shao LW, Pan Y, Qi XL, Li YX, Ma XL, Yi WN, Zhang J, Zhong
YF, Chang Q (2016) ATRX loss in adult supratentorial diffuse
astrocytomas correlates with p53 over expression and IDH1
mutation and predicts better outcome in p53 accumulated
patients. Histol Histopathol 31:103–114
22. Hewer E, Vajtai I, Dettmer MS, Berezowska S, Vassella E (2016)
Combined ATRX/IDH1 immunohistochemistry predicts genotype of oligoastrocytomas. Histopathology 68:272–278
23. Huang L, Jiang T, Yuan F, Li GL, Cui Y, Liu EZ, Wang ZC(2009) Correlation of chromosomes 1p and 19q status andexpressions of O6-methylguanine DNA methyltransferase(MGMT), p53 and Ki-67 in diffuse gliomas of World HealthOrganization (WHO) grades II and III: a clinicopathologicalstudy. Neuropathol Appl Neurobiol 35:367–379
24. Wakimoto H, Aoyagi M, Nakayama T et al (1996) Prognostic
significance of Ki-67 labelling indices obtained using MIB1
monoclonal antibody in patients with supratentorial astrocytomas. Cancer 77:373–380
25. Cai J, Yang P, Zhang C, Zhang W, Liu Y, Bao Z, Liu X, Du W,
Wang H, Jiang T, Jiang C (2014) ATRX mRNA expression
combined with IDH1/2 mutational status and Ki-67 expression
refines the molecular classification of astrocytic tumors: evidence
from the whole transcriptome sequencing of 169 samples samples. Oncotarget 5:2551–2561
26. Yan W, Zhang W, You G, Bao Z, Wang Y, Liu Y, Kang C,
You Y, Wang L, Jiang T (2012) Correlation of IDH1 mutation
with clinicopathologic factors and prognosis in primary123

Brain Tumor Pathol
glioblastoma: a report of 118 patients from China. PLoS One
7:e30339
27. Zeng A, Hu Q, Liu Y, Wang Z, Cui X, Li R, Yan W, Yo Y (2015)
IDH1/2 mutation status combined with Ki-67 labeling index
defines distinct prognostic groups in glioma. Oncotarget
6:30232–30238
28. Olar A, Wani KM, Alfaro-Munoz KD, Heathcock LE, van Thuijl
HF, Gilbert MR, Armstrong TS, Sulman EP, Cahill DP, VeraBolanos E, Yuan Y, Reijneveld JC, Ylstra B, Wesseling P,
Aldape KD (2015) IDH mutation status and role of WHO grade
and mitotic index in overall survival in grade II-III diffuse gliomas. Acta Neuropathol 129:585–596

123

29. Borodovsky A, Meeker AK, Kirkness EF, Zhao Q, Eberhart CG,
Gallia GL, Riggins GJ (2015) A model of a patient-derived IDH1
mutant anaplastic astrocytoma with alternative lengthening oftelomeres. J Neurooncol 121:479–487
30. England B, Huang T, Karsy M (2013) Current understanding of
the role and targeting of tumor suppressor p53 in glioblastoma
multiforme. Tumour Biol 34:2063–2074
31. Krell D, Mulholland P, Frampton AE, Krell J, Stebbing J, Bardella C (2013) IDH mutations in tumorigenesis and their potential
role as novel therapeutic targets. Future Oncol 9:1923–1935
32. Walerych D, Lisek K, Del Sal G (2015) Mutant p53: one, no one,and one hundred thousand. Front Oncol 5:289