Brain Tumor Pathology (2022) 39:14–24
https://doi.org/10.1007/s10014-021-00418-x

ORIGINAL ARTICLE

Reliability of IDH1‑R132H and ATRX and/or p53
immunohistochemistry for molecular subclassification of Grade 2/3
gliomasTomohide Nishikawa1 · Reiko Watanabe2 · Yotaro Kitano1,3 · Akane Yamamichi1 · Kazuya Motomura1 ·Fumiharu Ohka1 · Kosuke Aoki1 · Masaki Hirano1 · Akira Kato1 · Junya Yamaguchi1 · Sachi Maeda1 · Yuji Kibe1 ·Ryuta Saito1 · Toshihiko Wakabayashi1 · Yukinari Kato4 · Shuta Sato5 · Tomoyoshi Ogino5 · Atsushi Natsume1 ·Ichiro Ito5
Received: 31 August 2021 / Accepted: 11 November 2021 / Published online: 26 November 2021
© The Author(s) under exclusive licence to The Japan Society of Brain Tumor Pathology 2021

Abstract
Since the World Health Organization 2016 classification (2016 WHO), genetic status has been incorporated into the diagnosis of Grade 2/3 gliomas (lower-grade gliomas). Therefore, immunohistochemistry (IHC) of IDH1-R132H, ATRX, andp53 have been used in place of genetic status. We report the associations between histological findings, IHC, and genetic
status. We performed IHC of IDH1-R132H, ATRX, and p53 in 76 lower-grade gliomas and discussed its validity based on
the 2016 WHO and the upcoming 2021 WHO classification. The sensitivity and specificity of anti-ATRX, p53, and IDH1R132H IHC were 40.9%/98.1%, 78.6%/85.4%, and 90.5%/84.6%, respectively. Among 21 IDH1-mutant gliomas without
1p/19q codeletion, two gliomas (9.5%) mimicked the so-called classic for oligodendroglioma (CFO) in their morphology.
Of the 42 gliomas with 1p/19q codeletion, four cases were difficult to diagnose as oligodendroglioma through morphological examination. Moreover, there were three confusing cases with ATRX mutations but with retained ATRX-IHC positivity.
The lessons learned from this study are as follows: (1) ATRX-IHC and p53-IHC should be supplementary to morphologicaldiagnosis, (2) rare IDH mutations other than IDH1 R132H should be considered, and (3) there is no complete alternative
test to detect molecular features of glioblastoma under the 2021 WHO classification.
Keywords Glioma · Genetics · Immunohistochemistry · Surrogate marker · ATRX · p53 · WHO 5th CNS tumor
classification 2021

Introduction

Tomohide Nishikawa and Reiko Watanabe authors contributed
equally to this work.

The 2016 World Health Organization (WHO) classification of gliomas changed the way gliomas are classified[1]. Moreover, cIMPACT NOW and its updates 2–7 havebeen reported [2–8], which have added newly discovered2* Atsushi Natsume
anatsume@med.nagoya-u.ac.jp

Department of Pathology and Clinical Laboratories, National
Cancer Center Hospital East, 6‑5‑1 Kashiwanoha, Kashiwa,
Chiba 277‑8577, Japan3* Ichiro Itoichiro-ind@umin.ac.jp

Department of Neurosurgery, Mie University Schoolof Medicine, Tsu, Mie, Japan

4

Department of Molecular Pharmacology, Tohoku University
Graduate School of Medicine, Sendai, Miyagi, Japan

5

Department of Pathology, Nagano Red Cross Hospital,
5‑22‑1 Wakasato, Nagano, Nagano 380‑8582, Japan* Reiko Watanabe
reikwata@east.ncc.go.jp

1

Department of Neurosurgery, Nagoya University Schoolof Medicine, 65 Tsurumai, Showa, Nagoya, Aichi 466‑8550,
Japan

13

Vol:.(1234567890)

Brain Tumor Pathology (2022) 39:14–24

genetic alterations found in adulthood and childhood gliomas. These situations seem to have lessened the importanceof morphology-based histopathology. However, in routine
practice, pathologists are making efforts to evaluate the histology of a given tumor by summarizing limited information and diagnostic tools [9]. Histological evaluations are
particularly important in cases (1) requiring determination
within an urgent turn-around-time period, (2) with sample
insufficiency to perform accurate genetic analysis, and (3)in an institute where a molecular diagnostic technique is
not available.
Molecular diagnosis in Grades 2–4 gliomas focuses
mostly on the mutation of IDH1/2 and 1p/19q codeletion, although CDKN2A/B homozygous deletion, EGFR
amplification, TERT promoter (pTERT) mutation, mitosis,
and necrosis need to be considered for subclassification inprecision treatments for gliomas [2, 3, 5, 10]. The baseline
examination is the mutation of IDH1/2 and 1p/19q codeletion [11–13]. However, there are still controversial cases
as long as we use markers, IDH1-R132H, ATRX, and p53
immunohistochemistry (IHC). In this article, we investigated
how IHC-based diagnosis corresponds to the 2016 WHO
and upcoming 2021 WHO classifications [14] and discussed
their interpretation.

Materials and methods
Patients and data sets
We used the same tumor samples investigated in a previous study by Yamamichi et al. [15]. A total of 76 (45 maleand 31 female) patient tumor tissues were used for analysis
in this study, with informed consent from the patients, andethical approval from Nagoya University. The overall mean
age was 43.5 years old (Table 1). All tissues were obtained
between 2003 and 2012 by surgery and diagnosed as Grades
2/3 gliomas based on the 2016 WHO classification independently by two pathologists (R.W. and I. I.). When the
independent diagnosis was unmatched, a consensus diagnosis was made through discussion. The morphology withmicrovascular proliferations and necrosis that may be corresponded to glioblastoma (GBM), Grade 4 according to 2021
WHO was excluded in this study [14]. This study included
Grades 2 and 3 gliomas together as oligodendroglioma andastrocytoma. The role of molecular diagnostics has become
more weighted.

Tumor samples and analysis for molecular genetics
We examined 76 Grade 2 or 3 gliomas at Nagoya University Hospital between 2003 and 2012. The surgicallyremoved tumor tissue was first separated into two samples

15

for histology and molecular genetic analysis, as previously
described [15–17]. Fresh, unfixed, tumor tissue samples
were stored at – 80 °C and subsequently examined by wholeexome sequencing for IDH1/2, ATRX, TP53, pTERT, and by
SNP array for 1p/19q codeletion, CDKN2A/B homozygous
deletion, EGFR amplification, and chromosome 7 gain and
10 loss as was indicated in Yamamichi’s study [15].

Histology and IHC
Four slides from each case were freshly prepared fromformalin-fixed paraffin-embedded tumor tissues. One slide
was stained with hematoxylin and eosin (H&E), and theremaining three sections were used for immunostaining.
After deparaffinization, and dipping in 0.01 M phosphatebuffer saline at pH 7.2 for 10 min, the thin sections weretreated by antigen retrieval method using a citrate buffer
at pH 6.0 (BOND Epitope Retrieval Solution 1, AR9961,
Leica Biosystems) at 100 ℃ for 30 min for the first antibody H09 (DIA-H09, Dianova, Hamburg, dilution × 200)and HMab-2 [18, 19] (dilution × 1000), or using an ethylenediaminetetraacetic acid-based buffer at pH 9.0 (BOND
Epitope Solution 2, AR9640, Leica Biosystems) at 100 ℃
for 20 min for the first antibody HPA001906[19] (antibody
against ATRX, Sigma-Aldrich, dilution × 200), or at 100℃ for 30 min for the first antibody DO-7 [20] (antibody
against p53, Cell Signaling, dilution × 500). After rinsing
three times for 1–3 min with Bond Wash Solution (AR9590,
Leica Biosystems, Melbourne, Australia), each of the three
first antibodies indicated above were applied to a thin section of each sample. The first antibodies were diluted with
DAKO REAL™ Antibody Diluent (Agilent, Santa Clara,
CA, USA). After the first antibodies were reacted for 15 minat room temperature, washing steps were performed and peroxidase-conjugated antibodies were applied using a Leica
IHC Refine/NovoLink™ kit (Cat. DS9800), according to
the manufacturer’s instructions. All 76 specimens were
examined simultaneously using the same procedures by anautostainer machine (BOND-III Automated Stainer, Leica
Biosystems) and a detection kit (BOND-Polymer-RefineDetection kit, Leica Biosystems).
The sections were covered with a thin cover glass andsealing agent and microscopically examined by two pathologists (I. I. and R.W.). First, histological diagnoses were
performed using only H&E sections without genetic or
immunohistochemical information. Thereafter, immunohistochemical findings on each stained section were estimatedin the optimally stained portion of the tumor in each section.
According to “General Rules for Clinical and Pathological Studies on Brain Tumors, Fourth Edition” (2018, inJapanese), samples were judged as IHC-positive/negative forp53, ATRX, H09, or HMab-2 as follows: when 10% or more
neoplastic cells showed nuclear positivity for p53 (stronger

13

13

*C64
*C29
*C71
C24
C41
C75
*C03
C06
C12
*C15
C16
C21
C22
C25
C27
C28
C31
C32
C33
C34
C36
C37
C38
C39
C42
C47
C48
C51
C52
C53
C57
C61
C62
C67

Case
No

F
F
F
M
F
M
M
M
F
M
M
F
M
F
M
M
M
M
F
F
F
M
F
M
F
M
M
F
F
M
M
M
F
M

1R132H
2R172K
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H

Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos

Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y

IDH1 1p19q
R132H codeletion
IHC

IDH
genetics

42
30
44
29
55
38
56
56
33
54
53
44
66
31
33
57
50
41
40
56
30
30
72
44
54
28
64
56
33
28
51
58
45
35

1p19q

Age Sex IDH1/2

TP53

NonS
NonS
NonS
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype

Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos

Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype

Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
WildtypeATRX
ATRX TP53 genet- TP53 alteraalteration IHC
ics
tion

ATRX

Table 1  The results of genetic analyses and immunohistochemistry of the cases

Pos
Neg
Neg
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg

p53
IHC

Diagnosis

N
N
N
N
N
N
N
N
N
NonS SNV
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut
Mut

N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

N
N
N
N
N
N
N/A
N
N/A
N
N
N
N
N
N
N
N
N
N/A
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

O
O
O
O
O
O
A
O
O
A
O
O
O
O
O
O
O
O
O
O
O
O/A
O
O
O
O
O/A
O
O
O
O
O
O
O

O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O

2016WHO Provisional
2021WHOA (mut) OOOOOA (mut) O
A (mut) O
A (mut) O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O

CDKN2A/B pTERT EGFR + 7/-10 H&E IHCbased

Molecular features of GBM

16
Brain Tumor Pathology (2022) 39:14–24

C68
C70
C74
C77
C56
C58
C66
C72
C11
C43
C50
C55
C60
C54
C01
*C04
C05
C08
C09
*C10
C13
C78
C65
C26
C18
C30
C49
C76
C44
C35
C40
C59
C45
C20

Case
No

F
F
F
M
F
F
M
F
M
M
F
M
M
M
M
M
F
M
M
M
M
M
F
M
M
F
F
M
F
F
M
M
F
M

1R132H
1R132H
1R132H
1R132H
2R172K
1R132S
2R172K
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132H
1R132G
1R132H
1R132H
1R132H
1R132H
1R132S
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype

Pos
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Pos
Pos

Y
Y
Y
Y
Y
Y
Y
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

IDH1 1p19q
R132H codeletion
IHC

IDH
genetics

35
33
53
47
58
61
27
59
26
35
32
19
27
31
49
30
40
26
40
47
35
63
42
40
35
21
58
31
30
50
64
68
40
49

1p19q

Age Sex IDH1/2

Table 1  (continued)
TP53

Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
FSD
FSD
NonS
MM/S
SPL
FSI
FSD
NonS
StG SNVFSD
StG SNVSPL
StG SNV
FSD
SPL
FSD
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
StG SNVFSD
StG SNV
Wildtype
Wildtype

Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg

Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
2hit
2hit
2hit
1hit + LOH
1hit + LOH
1hit + LOH
2hit
1hit + LOH
2hit + LOH
2hit
1hit + LOH
1hit + LOH
3hit
2hit
1hit + LOH
1hit + LOH
1hit + LOH
1hit + LOH
1hit + LOH
1hit + LOH
1hit + LOH
1hit + LOH
2hit
Wildtype
2hit
1hit + LOH

Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
2 NonS
2 NonS
2 NonS
StG SNVNonS
StG SNV
2 NonSSPL
NonS + FSD
2 NonS
NonS
NonS
2NonS + FSD
2 NonS
StG SNVStG SNV
NonS
NonS
NonS
NonS
NonS
NonS
2NonS
Wildtype
2NonS
NonS

ATRX
ATRX TP53 genet- TP53 alteraalteration IHC
ics
tion

ATRX

Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Null
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Pos
Pos
Neg
Neg
Pos
Pos
Pos
Neg
Pos
Pos

p53
IHC

Diagnosis

N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Homodel
N
N

Mut
Mut
Mut
Mut
Mut
Mut
WT
Mut
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT

N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

N/A
N
N
N
N
N/A
N
N/A
N
N
N
N
N
N
N/A
N/A
N
N
N
N/A
N
N
N
N
N
N
N
N/A
N/A
N
N
N
N/A
N

O
O
O
O
O
O
O
O
A
A
A
A
A
O/A
A
O
A
A
A
O
A
A
A
A
A
A
TSm
A
A
A
O
O/A
A
A

O
O
O
O
O
O
O
O
A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (mut)A (wt)A (wt)A (wt)A (wt)A (wt)

2016WHO Provisional
2021WHO

O
O
O
O
O
O
O
O
A (wt) O
A (wt) O
A (wt) O
A (wt) O
A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)O
A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (mut) A (mut)A (wt) A (mut)A (mut) A (mut)A (mut) A (mut)O
A (mut)O
A (mut)A (wt) A (mut)A (wt) A (wt)
A (wt) A (wt)
A (wt) A (wt)A (mut) A (wt)A (mut) A (wt)

CDKN2A/B pTERT EGFR + 7/-10 H&E IHCbased

Molecular features of GBM

Brain Tumor Pathology (2022) 39:14–24
17

13

IHC immunohistochemistry, Histological diag. H&E histological diagnosis only on H&E, Y Yes, N No, pos. positive, neg. negative, NonS nonsynonymous, FSD frame shift mutation, FSI
frameshift insertion, MM/S multi mutant/splicing, StG stopgain, SNV single nucleotide variation, SPL splicing, LOH loss of heterozygosity, homodel homozygous deletion, Mut pTERT mutant,
WT pTERT wildtype, N/A not available, Null null staining, A astrocytoma, A (mut) astrocytoma, IDH-mutant, A (wt) astrocytoma, IDH-wildtype, O oligodendroglioma, GBM Glioblastoma, O/A
difficult to distinguish O and A, TSm Too small for diagnosis

The data were sorted in the order of IDH1/2 mutation (mutated or not), 1p19q codeletion (codeleted or not), TP53 mutation (mutated or not), and ATRX mutation (mutated or not)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)A (wt)
O
A
A
A
A
A
O
A
N
N
N/A
N/A
N
N
N
N
N
N
N
amp
N
N
N
N
WT
Mut
WT
Mut
Mut
WT
WT
Mut
N
N
N
N
N
N
N
N
Pos
Pos
Neg
Pos
Pos
os
Neg
Neg
2NonS + FSD
StG SNV
FSD
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
3hit
1hit + LOH
1hit
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
N
N
N
N
N
N
N
N
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
Wildtype
M
M
F
M
M
M
F
M
26
53
66
42
51
41
49
40
C07
C17
C02
C14
C23
C63
C69
C73

IDH1 1p19q
R132H codeletion
IHC
IDH
genetics

ATRX
ATRX TP53 genet- TP53 alteraalteration IHC
ics
tion

p53
IHC

CDKN2A/B pTERT EGFR + 7/-10 H&E IHCbased

Diagnosis
Molecular features of GBM
TP53
ATRX
1p19q
Age Sex IDH1/2

Case
No

Table 1  (continued)

13

A (wt)GBM
A (wt)GBM
GBM
A (wt)A (wt)
GBM

Brain Tumor Pathology (2022) 39:14–24
2016WHO Provisional
2021WHO

18

than wild-type staining on adjacent non-neoplastic cells), it
was considered IHC-positive; when neoplastic cells showedno nuclear staining in ATRX IHC in comparison with clear
positivity of adjacent non-neoplastic cells, it was considered IHC-negative for ATRX; when neoplastic cells showed
nuclear and cytoplasmic positivity for H09 or HMab-2, it
was considered IHC-positive.
Morphologically, oligodendroglioma was diagnosed
when a given case showed a typical perinuclear halo withno cytoplasmic process, namely, classic for oligodendroglioma (CFO) [21]. On the contrary, if a tumor had evident
cell processes and a wide cytoplasm, the morphologicaldiagnosis was astrocytoma. Even if a given tumor cell had
some amount of cytoplasm, it was diagnosed as oligodendroglioma when small round nuclei were observed in a monotonous manner.

Results
Table 1 indicates each patient’s age, sex, IDH1/2 mutation
status and H09-IHC positivity, biallelic T53 genetic status
and DO-7-IHC positivity, GBM molecular features such as
CDKN2A/B homozygous deletion, pTERT mutation, EGFR
amplification, and both chromosome 7 gain and chromosome 10 loss, and diagnose based on only H&E staining,
integrated IHC, 2016 WHO classification, or provisional
diagnosis based on the upcoming 2021 WHO classification. First, we display morphologically confusing cases anddemonstrate the differences between genetic testing and IHC
for IDH, ATRX and TP53 status, sensitivity and specificityof ATRX and/or p53 IHC as surrogate markers for 1p/19q
codeletion. Finally, we show the association of IHC-based
integrated diagnosis with molecular genetic diagnosis basedon the 2016 WHO and upcoming 2021 WHO classifications.

Morphology
We attempted to morphologically achieve a preliminary
diagnosis using H&E sections. As shown in the column for“H&E” in Table 1, most cases were consistent with” IHCbased” and “WHO2016-based” diagnoses. However, there
were some confusing cases (* C No. in Table 1). There were
three oligodendrogliomas with 1p/19q codeletion, but withretained ATRX immunopositivity despite having ATRX andpTERT mutation, and the wild-type TP53 as what Yamamichi et al. have already reported [15]: two cases (Cases 64
and 71) had ATRX nonsynonymous mutation and showed
many tumor cells with spindle-shaped cytoplasm and bipolar
processes (Fig. 1a–d). The remaining case (Case 29) did nothave a spindle-shaped cell morphology but had histology
conventional to oligodendroglioma (Fig. 1e, f). Notably, case29 had an IDH2R172K mutation.

Brain Tumor Pathology (2022) 39:14–24

Cases 3 and 15 were genetically oligodendrogliomas,
with 1p/19q codeletion, IDH1R132H mutation, and ATRX
wild-type, TP53 wild-type, and pTERT mutation. However,
the tumor cells of Case 3 showed small round or oval nuclei,
eosinophilic cytoplasm, and bipolar processes without aperinuclear halo (Fig. 2a). The tumor cells of Case 15 hadirregularly shaped nuclei, some of which showed short spindles or irregular shapes (Fig. 2b).
Likewise, astrocytomas with typical genetic alterations,
including mutated IDH1R132H, 1p/19q non-codeleted,mutated ATRX, and mutated TP53 (Cases 4 and 10), showedoligodendroglioma-like morphology; the tumor cells had
irregularly shaped round nuclei with a perinuclear halo
(Fig. 2c, d).

IDH1‑R132H IHC
We determined the sensitivity and specificity of two worldwide anti-IDH1 R132H antibodies: H09 and HMab-2.
The sensitivity and specificity of H09 for detecting IDH1
R132H were 98.2% and 84.2%, respectively, while thoseof HMab-2 were 89.5% and 52.6%, respectively (Table 2).
H09 was more sensitive and specific than HMab-2; however, even with H09, among the 57 gliomas with genetic
IDH1R132H, one (Case 72) failed to be detected by IDH1R132H-IHC (H09). In addition, the antibody was positivein 2 out of 13 IDH1/2-wild type tumors (Cases 45 and 20,
15.4%) (Table 1). As for real-world sensitivity and specificity on H09, the number of false negative cases may include
other mutations than R132H. From this standpoint, IDH IHC
using H09 detected correctly 57 cases out of all kinds of
IDH1/2 mutations in our cases and detected falsely 2 cases
out of IDH1/2 wild type 13 cases. These data gave sensitivity of 90.5% and specificity of 84.6% on H09 IHC (notincluded in Table 2).

ATRX mutation
ATRX alteration status included multiple mutations, splicing, frameshift insertion/deletion, nonsynonymous, andstop-gain single nucleotide variants (Table 1). These mutational statuses did not correlate with retained or lost ATRX
immunopositivity, resulting in low sensitivity for ATRXIHC (40.9%). However, most ATRX wild-type cases showedretained ATRX positivity (specificity of 98.1%) (Table 3A).

TP53 mutation
TP53 had a variety of alterations such as biallelic nonsynonymous mutation, frameshift deletion, frameshift insertion,
multi-mutant/splicing, stop-gain single nucleotide variants,
and splicing (Table 1). The sensitivity and specificity of p53

19

IHC against TP53 mutations were 78.6% and 85.4%, respectively (Table 3C).

Sensitivity and specificity of surrogate markers
Diffuse astrocytoma, IDH-mutant, is most likely to harbormutations in ATRX and TP53; therefore, this tumor typetheoretically displays loss of ARTX immunopositivity andretains p53 positivity. However, when evaluating whether
they serve as a surrogate marker for 1p/19q codeletion, thesensitivity and specificity of ATRX IHC were 100% and29.4%, respectively (Table 3B). Similarly, when we compared p53 IHC negativity with 1p/19q codeletion, the sensitivity and specificity for 1p/19q codeletion status were 90.5%
and 73.5%, respectively (Table 3D). Therefore, ATRX and
p53 IHC may work complimentarily as surrogate markers
for 1p/19q codeletion.
When we combined “ATRX-retained and p53 wild-type”
IHC results as a surrogate marker, the specificity for 1p/19q
codeletion increased to 76.5%, and the sensitivity remained
at 90.5% (Table 3E). Figure 3 shows the flow of IHC-based
diagnosis: among 76 cases, 17 cases were H09-negative;
they were categorized as diffuse astrocytoma, IDH-wildtype. In fact, there were four oligodendrogliomas, IDHmutant with 1p/19 codeletion, and two diffuse astrocytomas, IDH-mutant, according to the 2016 WHO classification
system. When ATRX was positive and p53 was negative, thetumors were diagnosed as oligodendroglioma, IDH-mutant,and 1p/19q codeletion. In our cohort, 37 patients were ofthis type. Genetic testing revealed that the three tumors
were non-codeleted. Diffuse astrocytoma, IDH-mutant,
was diagnosed when ATRX was negative, AND/OR p53
was positive. Among the 22 cases meeting these criteria,
four were actually oligodendroglioma, IDH-mutant with
1p/19q codeletion, and two were diffuse astrocytoma, IDH
wild-type according to the 2016 WHO scheme. Each caseis shown in Table 1.

Provisional molecular diagnosis basedon the upcoming 2021 WHO classification
The 2021 WHO classification is upcoming as of November, 2021. We made a provisional molecular diagnosis
according to the 2021 WHO classification, as shown in
Table 1. Three cases (Cases 17, 23, and 73) had pTERT
mutation, and one case (Case 14) had pTERT mutationand EGFR amplification, and thus these were classified
as GBM. Case 59 with wild-type IDH had a CDKN2A/B
homozygous deletion. However, it was designated as astrocytoma, IDH wild-type.

13

20

Brain Tumor Pathology (2022) 39:14–24

Fig. 1  Representative caseswith discrepancies between
morphology and immunohistochemistry. Three gliomas areshown which have discrepancies
between the ATRX molecularanalysis and IHC results. They
have 1p19q codeletion, IDH
mutation, and ATRX mutation(cases 64 (a, b), 71 (c, d), and29 (e, f)). In case 64 (a) and 71(c), the tumor cells have small
oval or short spindle nuclei
with narrow, long, pale, orwide eosinophilic cytoplasmic
processes and almost no perinuclear halo. ATRX immunohistochemistry (IHC) shows
clear nuclear positivity (b andd), despite the non-synonymous
ATRX mutation. In case 29 (e),tumor cells are small, round,or oval-shaped nuclei with aperinuclear halo. This case has
IDH2R172K mutation and nonsynonymous ATRX mutation,
whereas ATRX IHC is also
positive (f). Scale bar, 20 μm

Discussion
The diagnosis of lower-grade glioma has changed from amorphology-oriented system to a genetic-event-oriented
system since the WHO 2016 classification. Thereby, theprognosis of the disease can be predicted more accurately
based on genetic data than on morphology [15]. However, as
it would be difficult to fully analyze the genetics of all cases,
some immunohistochemical surrogate markers are required.
The aim of this study was to investigate the reliability of surrogate markers, that is, ATRX- and p53-IHC, by evaluating
the patterns of ATRX alteration and TP53 mutation in nextgeneration sequencing (NGS) and classical morphology. The
lessons learned from this study are as follows: (1) ATRXIHC and p53-IHC should be supplementary to morphological diagnosis, (2) the possibility of rare IDH mutations otherthan IDH1 R132H should be considered, and (3) there is no13

easy alternative test to detect molecular features of GBM
under the 2021 WHO classification.
The cIMPACT-NOW Update 2 states that a definite lossof ATRX nuclear expression and/or strong, diffuse p53
immunopositivity is sufficient for the diagnosis of astrocytoma without the need for a 1p/19q test [3]. Sonoda et al.
developed a practical procedure in which immunohistochemistry was used as the next step after a simple, but notrecommended, diagnosis by pathology alone, and ATRX
and p53 IHC could be used as a surrogate for 1p/19q [22].
Our data support these reports by additionally providing
the feasibility and reliability of combined ATRX and p53
IHCs in clinical practice.
Few studies have compared potential IHC surrogates andgenetic analysis in Grade 2/3 gliomas, while some reports
have suggested that the presence of ATRX mutations could
be a surrogate marker for 1p/19q codeletion [23–25]. Thesestudies limited the utility of immunohistochemistry toBrain Tumor Pathology (2022) 39:14–24

21

Fig. 2  Four gliomas suggesting discrepancies between morphology
and 1p/19q status. In Case 3 (a), the tumor cells show little or no
perinuclear halo, small but irregularly shaped nuclei, and eosinophilic
cytoplasmic processes such as astrocytoma. In Case 15 (b), tumor
cells have cytoplasmic processes and show nuclear pleomorphism,occasionally binucleated, seemingly astrocytoma. Both tumors have
IDH1R132H mutants, ATRX-wild type, and 1p/19q codeletion. In

Case 4 (c), a honeycomb pattern is observed, but it has mutations inboth IDH1R132H and ATRX and 1p/19q non-codeleted, that is, alterations compatible with astrocytoma. In Case 10 (d), tumor cells havesmall or medium-sized, round nuclei, with some having a perinuclear
halo; however, this case has neither 1p/19q codeletion nor IDH mutation. Scale bar, 20 mm

Table 2  The results of IDH1-R132H-immunostaining using antibodies, H09 and HMab-2
H09

IDH IHC positive
IDH IHC negative

IDH1 R132H

Hmab-2

IDH1
R132H
mutatedIDH1 R132H wildtype

56
1
57

3
16
19
Sensitivity
Specificity

59
17
98.2%
84.2%

provide genetic information on tumors. Hewer et al. statedthat ATRX-proficient tumors with IDH1 IHC positivity
predicted 1p/19q loss of heterozygosity with a sensitivityof 85% [23]. Rajeswarie et al. classified gliomas according
to their immunohistochemical surrogate markers. However,their studies did not provide a genetic status using NGS [26].
There seems to be an incomplete parallel relationshipbetween mutational status and IHC results for ATRX,IDH IHC positive
IDH IHC negative

IDH1 R132H
IDH1
R132H
mutatedIDH1 R132H wildtype

51
6
57

9
10
19
Sensitivity
Specificity

60
16
89.5%
52.6%

IDH, and TP53. The IHC results may be susceptible tothe examination conditions. It is difficult to set the optimal
conditions for IHC staining and to estimate the results.
Therefore, the sensitivity and specificity may be affectedby inter-laboratory and inter-condition variations. In ourstudy, the cohort was collected from a single institute andanalyzed retrospectively. When archived paraffin-embedded tissues are used, one must consider whether the antigenicity is well-preserved [27, 28].

13

22

Brain Tumor Pathology (2022) 39:14–24

Table 3  Sensitivity and specificity of ATRX IHC for ATRX mutation (A), for 1p/19q codeletion (B), p53 IHC for TP53 mutation (C), for 1p/19q
codeletion (D) and combination of ATRX IHC and p53 IHC for 1p/19q codeletion (E)(A)

ATRX IHC negative
ATRX IHC positive(C)

p53 IHC positive
p53 IHC negative(E)

ATRX IHC positiveand p53 IHC negative
ATRX IHC negative
and/or p53 IHC
positive

ATRX

(B)

ATRX mutated

ATRX wildtype

9
13
22

1
53
54
Sensitivity
Specificity

1p/19q
1p/19q
1p/19q non-codeleted
codeleted

1066

ATRX IHC positive
ATRX IHC negative

40.9%
98.1%

TP53

(D)

TP53 mutated

TP53 wildtype

22
6
28

7
41
48
Sensitivity
Specificity

42
0
42

24
10
34
Sensitivity
Specificity

66
10
100.0%
29.4%

1p/19q
1p/19q
1p/19q non-codeleted
codeleted

2947

p53 IHC negative
p53 IHC positive

78.6%
85.4%

38
4
42

9
25
34
Sensitivity
Specificity

47
29
90.5%
73.5%

1p/19q
1p/19q codeleted

1p/19q non-codeleted

38

8

46

4

26

30

42

34
Sensitivity
Specificity

90.5%
76.5%

Fig. 3  Comparison amongclassifications of lower-grade
gliomas according to immunohistochemistry for IDH1R132H, ATRX, and/or p53.
IDH1-R132H IHC (H09)-based
diagnosis leads to 17 “diffuse
astrocytoma (DA), IDH-wildtype” diagnoses, among which
there are six genetic IDH1/2
mutant cases. ATRX and p53
IHC-based diagnosis leads to
37 “oligodendroglioma (OD),
IDH-mutant, 1p/19q codeleted”
and 22 “diffuse astrocytoma,
IDH-mutant” diagnoses. However, there are 3 and 6 casesthat differed from the genetic
diagnosis, respectively

IDH1 R132H IHC
Positive: 59

Negative: 17

ATRX IHC / p53 IHCATRX negative
and/orp53 positive:22

ATRX positiveandp53 negative:37
estimated as OD, IDH mutant and1p/19qcodeleted

34

3

estimated as DA, IDH mutant

4

16

2

estimated as DA, IDH wild-type

4 2

11

OD, IDH mutant and 1p/19q codeleted
DA, IDH mutant
DA, IDH wild-type
actual diagnosis according to WHO 201613

Brain Tumor Pathology (2022) 39:14–24

We demonstrated that the specificity and sensitivity
for 1p/19q codeletion were higher (76.5%/90.5%) whencombined with ATRX-IHC retention and p53-IHC wild
pattern (Table 3E). Many sought other surrogates; Rajmohan et al. reported that alpha internexin is a surrogate
marker for 1p/19q codeletion, with a specificity of 93.0%
and a sensitivity of 87.5% [29]. Kitahama et al. reported
that H3K27me3 loss in IHC tended to be a 1p/19q surrogate with high specificity but low sensitivity that was notobserved in oligodendrogliomas with IDH mutations otherthan IDH1-R132H [30]. However, the underlying mode ofaction remains unknown in these studies and they provide
only circumstantial evidence.
The pathological diagnosis began with H&E-based
morphological observations, which correctly determined
38 cases of 42 genetic oligodendrogliomas and 26 casesof 34 genetic astrocytomas. However, there are a substantial number of cases that could not be diagnosed correctly
using only H&E-based morphological examination, as
depicted in Figs. 1 and 2. Integrated with IHC and morphology, the correct diagnosis could be achieved in mostcases.
IHC- and morphology-based diagnosis has, however, alimitation. In our cohot, 63 IDH-mutant gliomas harbored
six IDH1/2 mutations other than IDH1-R132H. It would
be hazardous to determine the IDH mutation status using
only IDH1-R132H IHC. Regardless of the tumor grading,
IDH-wild type astrocytoma can be classified as a GBMlike entity; thus, patients require intensive radiochemotherapy. This study suggested that there were 15.4% false
positives and 9.5% false negatives for IDH1/2 mutations.
This error rate would raise a new concern that patients
may receive over- or under-treatment when 2021 WHO
is applied.
Acknowledgements This research was supported by a Grant-in-Aid for
Scientific Research on Innovative Areas “Chemistry for Multimolecular
Crowding Biosystems” (A.N.) (JSPS KAKENHI Grant No. 17928985).
This research was also supported in part by the Japan Agency for
Medical Research and Development (AMED) under Grant Number:
JP21am0101078 (to Y.K.).
Author contributions Conceptualization: A.N., R.W. and I.I.; Methodology: A.N., A.Y., K.M., Y. Kitano, R.W., and I.I.; Conduct of Immunohistochemistry: S.S. and T.O.; Formal analysis and investigation:
Y. Kitano, A.Y. S.M., Y.K., A.K., R.S., T. W., R.W., and I.I.; Writingoriginal draft preparation: T.N., R.W., and I.I.; Writing-review andediting: R.W., I.I., and A.N.; Funding acquisition: A.N. and Y. Kato;
Resources: Y.M., F.O., K.A., M.M., J.Y., Y. Kitano and Y. Kato; Supervision: A.N. and I.I.
Funding Japan Society for the Promotion of Science,17928985,Atsushi
Natsume,Japan Agency for Medical Research and Development,JP21am0101078,Yukinari Kato

23

References
1. Louis DN, Perry A, Reifenberger G et al (2016) The 2016 World
health organization classification of tumors of the central nervous
system: a summary. Acta Neuropathol 131:803–820
2. Louis DN, Wesseling P, Paulus W et al (2018) cIMPACT-NOW
update 1: not otherwise specified (NOS) and not elsewhere classified (NEC). Acta Neuropathol 135:481–484
3. Louis DN, Giannini C, Capper D et al (2018) cIMPACT-NOW
update 2: diagnostic clarifications for diffuse midline glioma, H3
K27M-mutant and diffuse astrocytoma/anaplastic astrocytoma,
IDH-mutant. Acta Neuropathol 135:639–642
4. Brat DJ, Aldape K, Colman H et al (2018) cIMPACT-NOW
update 3: recommended diagnostic criteria for “Diffuse astrocytic
glioma, IDH-wildtype, with molecular features of glioblastoma,
WHO grade IV.” Acta Neuropathol 136:805–810
5. Ellison DW, Hawkins C, Jones DTW et al (2019) cIMPACT-NOW
update 4: diffuse gliomas characterized by MYB, MYBL1, or
FGFR1 alterations or BRAF(V600E) mutation. Acta Neuropathol
137:683–687
6. Brat DJ, Aldape K, Colman H et al (2020) cIMPACT-NOW
update 5: recommended grading criteria and terminologies for
IDH-mutant astrocytomas. Acta Neuropathol 139:603–608
7. Louis DN, Wesseling P, Aldape K et al (2020) cIMPACT-NOW
update 6: new entity and diagnostic principle recommendations of
the cIMPACT-Utrecht meeting on future CNS tumor classificationand grading. Brain Pathol 30:844–856
8. Ellison DW, Aldape KD, Capper D et al (2020) cIMPACT-NOW
update 7: advancing the molecular classification of ependymal
tumors. Brain Pathol 30:863–866
9. Paulus W (2017) WHO 2016: Open questions and practical implications. Acta Neurochir (Wien) 159:419–422
10. Pekmezci M, Rice T, Molinaro AM et al (2017) Adult infiltrating
gliomas with WHO 2016 integrated diagnosis: additional prognostic roles of ATRX and TERT. Acta Neuropathol 133:1001–1016
11. Gorovets D, Kannan K, Shen R et al (2012) IDH mutation andneuroglial developmental features define clinically distinct subclasses of lower grade diffuse astrocytic glioma. Clin Cancer Res
18:2490–2501
12. Kefenie H, Desta B, Abebe A et al (1989) Prevalence of hepatitis
B infection among hospital personnel in Addis Ababa (Ethiopia).
Eur J Epidemiol 5:462–467
13. Cairncross G, Wang M, Shaw E et al (2013) Phase III trial ofchemoradiotherapy for anaplastic oligodendroglioma: long-term
results of RTOG 9402. J Clin Oncol 31:337–343
14. Louis DN, Perry A, Wesseling P et al (2021) The 2021 WHOclassification of tumors of the central nervous system: a summary.
Neuro Oncol 23:1231–1251
15. Yamamichi A, Ohka F, Aoki K et al (2018) Immunohistochemical
ATRX expression is not a surrogate for 1p19q codeletion. Brain
Tumor Pathol 35:106–113
16. Suzuki H, Aoki K, Chiba K et al (2015) Mutational landscapeand clonal architecture in grade II and III gliomas. Nat Genet
47:458–468
17. Aoki K, Natsume A (2019) Overview of DNA methylation in adult
diffuse gliomas. Brain Tumor Pathol 36:84–91
18. Kato Y (2015) Specific monoclonal antibodies against IDH1/2
mutations as diagnostic tools for gliomas. Brain Tumor Pathol
32:3–11
19. Fujii Y, Ogasawara S, Oki H et al (2015) A high-sensitive HMab-2
specifically detects IDH1-R132H, the most common IDH mutation in gliomas. Biochem Biophys Res Commun 466:733–739
20. Takami H, Yoshida A, Fukushima S et al (2015) Revisiting TP53
mutations and immunohistochemistry–a comparative study in 157
diffuse gliomas. Brain Pathol 25:256–265

13

24
21. Giannini C, Burger PC, Berkey BA et al (2008) Anaplastic oligodendroglial tumors: refining the correlation among histopathology, 1p 19q deletion and clinical outcome in Intergroup Radiation
Therapy Oncology Group Trial 9402. Brain Pathol 18:360–369
22. Sonoda Y, Yokoo H, Tanaka S et al (2019) Practical proceduresfor the integrated diagnosis of astrocytic and oligodendroglial
tumors. Brain Tumor Pathol 36:56–62
23. Hewer E, Vajtai I, Dettmer MS et al (2016) Combined ATRX/
IDH1 immunohistochemistry predicts genotype of oligoastrocytomas. Histopathology 68:272–278
24. Takano S, Ishikawa E, Sakamoto N et al (2016) Immunohistochemistry on IDH 1/2, ATRX, p53 and Ki-67 substitute molecular
genetic testing and predict patient prognosis in grade III adult
diffuse gliomas. Brain Tumor Pathol 33:107–116
25. Leeper HE, Caron AA, Decker PA et al (2015) IDH mutation,
1p19q codeletion and ATRX loss in WHO grade II gliomas.
Oncotarget 6:30295–30305
26. Rajeswarie RT, Rao S, Nandeesh BN et al (2018) A simple algorithmic approach using histology and immunohistochemistry for

13

Brain Tumor Pathology (2022) 39:14–24

27.
28.
29.

30.

the current classification of adult diffuse glioma in a resourcelimited set-up. J Clin Pathol 71:323–329
Grillo F, Bruzzone M, Pigozzi S et al (2017) Immunohistochemistry on old archival paraffin blocks: is there an expiry date? J Clin
Pathol 70:988–993
Combs SE, Han G, Mani N et al (2016) Loss of antigenicity withtissue age in breast cancer. Lab Invest 96:264–269
Rajmohan KS, Sugur HS, Shwetha SD et al (2020) Alphainternexin: a surrogate marker for 1p/19q codeletion and prognostic marker in anaplastic (WHO grade III) Gliomas. Neurol
India 68:832–837
Kitahama K, Iijima S, Sumiishi A et al (2021) Reduced
H3K27me3 levels in diffuse gliomas: association with 1p/19q
codeletion and difference from H3K27me3 loss in malignant
peripheral nerve sheath tumors. Brain Tumor Pathol 38:23–29

Publisher's Note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliations.