Brain Tumor Pathology (2018) 35:106–113
https://doi.org/10.1007/s10014-018-0312-5

ORIGINAL ARTICLE

Immunohistochemical ATRX expression is not a surrogate for 1p19q
codeletion
Akane Yamamichi1 · Fumiharu Ohka1 · Kosuke Aoki1 · Hiromichi Suzuki1 · Akira Kato1 · Masaki Hirano1 ·Kazuya Motomura1 · Kuniaki Tanahashi1 · Lushun Chalise1 · Sachi Maeda1 · Toshihiko Wakabayashi1 · Yukinari Kato2 ·Atsushi Natsume1
Received: 23 February 2018 / Accepted: 7 March 2018 / Published online: 17 March 2018
© The Japan Society of Brain Tumor Pathology 2018

Abstract
The IDH-mutant and 1p/19q co-deletion (1p19q codel) provides significant diagnostic and prognostic value in lower-grade
gliomas. As ATRX mutation and 1p19q codel are mutually exclusive, ATRX immunohistochemistry (IHC) may substitute for
1p19q codel, but this has not been comprehensively examined. In the current study, we performed ATRX-IHC in 78 gliomas
whose ATRX statuses were comprehensively determined by whole exome sequencing. Among the 60 IHC-positive and 18
IHC-negative cases, 86.7 and 77.8% were ATRX-wildtype and ATRX-mutant, respectively. ATRX mutational patterns were
not consistent with ATRX-IHC. If our cohort had only used IDH status and IHC-based ATRX expression for diagnosis, 78
tumors would have been subtyped as 48 oligodendroglial tumors, 16 IDH-mutant astrocytic tumors, and 14 IDH-wildtype
astrocytic tumors. However, when the 1p19q codel test was performed following ATRX-IHC, 8 of 48 ATRX-IHC-positive
tumors were classified as “1p19q non-codel” and 3 of 16 ATRX-IHC-negative tumors were classified as “1p19q codel”; atotal of 11 tumors (14%) were incorrectly classified. In summary, we observed dissociation between ATRX-IHC and actual
1p19q codel in 11 of 64 IDH-mutant LGGs. In describing the complex IHC expression of ATRX somatic mutations, ourresults indicate the need for caution when using ATRX-IHC as a surrogate of 1p19q status.
Keywords Glioma · ATRX · Immunohistochemistry · Whole exome sequencing · 1p19q codel

Introduction
Gliomas are common brain tumors. Although grade II and
III gliomas (lower-grade gliomas, LGGs) are less aggressive than glioblastoma multiforme, most result in recurrence
and eventual fatality [1, 2]. In the revised 4th edition of theclassification of tumors of the central nervous system of theWorld Health Organization in 2016, in addition to histology, genetic markers were included to define tumor entities[3]; accordingly, the status of the IDH mutation and 1p19q* Atsushi Natsume
anatsume@med.nagoya‑u.ac.jp
1

Department of Neurosurgery, Nagoya University
School of Medicine, 65 Tsurumai‑cho, Showa‑ku,
Nagoya 466‑8550, Japan2

Department of Antibody Drug Development, Graduate
School of Medicine, New Industry Creation Hatchery Center,
Tohoku University, Sendai, Miyagi, Japan

13

Vol:.(1234567890)

codeletion (1p19q codel) became essential for the diagnosis
and prognosis of gliomas, particularly for LGGs [4–8].
A variety of methods [9] for analyzing the 1p19q codelsuch as fluorescence in situ hybridization [6, 7, 10], comparative genomic hybridization [11], and multiplex ligationdependent probe amplification [12] have been developed,
but have limitations in routine clinical practice because of
their complexity and cost. Accordingly, molecular surrogate
markers for the 1p19q codel are needed for LGGs.
ATRX mutation is thought to occur at an early stage ofglioma tumorigenesis [1] and influence its development[13–15], thereby playing a potential role as a glioma classification marker. However, ATRX mutation shows various
patterns (i.e., non-recurrent mutations) [16] and sequencing is complicated and time-consuming. ATRX mutationand 1p19q codel are mutually exclusive [1, 8]. On the other
hand, the presence of ATRX-wildtype does not necessarily
indicate the presence of 1p19q codel (Table 1). However,assuming that ATRX mutation prevents the expression of
ATRX [17], ATRX negativity in immunohistochemistry

Brain Tumor Pathology (2018) 35:106–113
Table 1  Mutual exclusivenessof 1p/19q codeletion and ATRX
mutation in three cohorts

107
Current study
1p19q
codel
(+)

JPN

1p19q codel (−)

Grades II/III gliomas
ATRX-mutant
3
19
ATRX-wildtype 40
16
IDH-mutant grade II/III gliomas
ATRX-mutant
3
16
ATRX-wildtype 40
5
p < 0.00001

TCGA​

1p19q
codel
(+)

1p19q codel (−)

1p19q
codel
(+)

1p19q codel (−)

5
143

91
96

3
135

157
127

5
143

85
28
p < 0.00001

3
135

156
48
p < 0.00001

Each genetic status was determined as described previously [1, 26, 27]
JPN Japanese Cohort, TCGA​The Cancer Genome Atlas Cohort(IHC) is a possible substitute for detection of 1p19q codel.
Some studies demonstrated that ATRX immunonegativity
in IHC coincided with ATRX mutation [18–23]. Moreover,some reports proposed that ATRX-IHC should be used forclinical classification without assessment of the 1p19q codel[19, 23–25]. However, few reports have examined the sensitivity and specificity of ATRX-IHC for ATRX mutation and
1p19q codel in a relatively large cohort.
In this study, we investigated whether ATRX loss in IHC
is consistent with ATRX mutation and 1p19q codel identified
by whole-exome sequencing (WES) in 78 glioma samples.

Materials and methods
Mutual exclusivity of ATRX mutation and 1p19q
codel in two large cohorts
To investigate mutual exclusivity of ATRX mutation and
1p19q codel, we used data sets from two independentcohorts of LGG patients from Japan (JPN) (n = 335) and
The Cancer Genome Atlas (TCGA, n = 422) [26] (Table 1).
Mutations in IDH1, IDH2, and ATRX were investigatedusing WES and/or targeted deep sequencing of custom bait
library or PCR-amplified fragments as previously described.
The 1p19q codel status was determined, as described previously [1, 27].

Tumor samples and gene mutation analysis
All 78 patients were diagnosed with grade II or III gliomas independently by three pathologists. With informed
consent, all tumor samples were collected at the time ofsurgery at Nagoya University between 2003 and 2012. All
ATRX-mutant samples were collected during initial surgery
without pretreatment. WES of these samples was performed
as part of a large cohort in a previous report [1]. Mutationsin IDH1/2 and ATRX and the 1p19q codel were analyzed
using deep sequencing for all samples.

Immunohistochemistry
For the primary reaction, two anti-ATRX antibodies andan anti-IDH1 R132H antibody were used for immunohistochemistry. Because ATRX is expressed in normal cells,
to avoid incorrect assignment of ATRX immunopositivity to non-tumor cells, adjacent section was stained using
an anti-IDH1-R132H antibody. AMab-6 (Wako, Osaka,
Japan) [28] is a monoclonal anti-ATRX antibody producedin mice and HPA001906 is a polyclonal anti-ATRX antibody(Sigma-Aldrich, St. Louis, MO, USA) produced in rabbits.
HMab-2 (Wako) [29] is an anti-IDH1 R132H monoclonal
antibody produced in mice. The tumor samples were fixedwith 10% formalin and embedded in paraffin. Sections(5-µm thick) were prepared using a microtome (REM710,
Yamato Kohki Industrial, Saitama, Japan). After deparaffinization and hydration, the sections were incubated in
retrieval solution (Tris-EDTA buffer pH 9.0 for HMab-2
and HPA001906, and citrate acid buffer pH 6.0 for AMab6) for 20 min at 100 °C in an electric pod. After antibody
retrieval, subsequent procedures were performed using aHistostainer automatic immunostaining machine (HS36A,
Nichirei Bioscience Inc., Tokyo, Japan). The sections were
blocked with antibody diluent (Dako; Agilent Technologies,
Inc., Santa Clara, CA, USA) at room temperature for 10 min
and incubated for 1 h with each primary antibody diluted
to 1 µg/ml. An appropriate second labeled polymer fromthe EnVision HRP kit (Dako) was used and the sections
were incubated for 30 min. The Histostainer was used until
incubation of second antibody and subsequent washing, andsubstrate-chromogen solution from the DAB Substrate Kit(Vector Laboratories, Burlingame, CA, USA) was applied
for 10 min. After washing, the sections were counter-stained

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108

with hematoxylin and mounted in Multi-Mount (Matsunami
Glass Inc., Kishiwada, Japan).

Multiple fluorescence immunohistochemistry
We carefully classified the tumors by staining ATRX andIDH1-R132H in 2 consecutive sections or used multicolored co-immunofluorescent histology to confirm that
ATRX expression was derived from tumor cells, rather than
ATRX-positive normal cells. Fluorescence IHC (fIHC)
was performed using the Opal 4-color Manual IHC kit(PerkinElmer, Inc., Waltham, MA, USA). HMab-2 and
HPA001906 were used as primary antibodies in the same
sections. A similar procedure as for normal immunohistochemistry was used until incubation and washing HMab-2
as the first primary antibody. After washing HMab-2, thesecondary antibody labeled HRP was applied and incubatedfor 10 min. After washing, Opal 570 reagent was applied andincubated for 10 min. After washing, the sections were reincubated in antigen retrieval solution for 15 min at 100 °C
in an electric pod with blocking. HPA001906 antibody wasapplied as a second primary antibody and incubated for 1 h.
After washing, a second polymer labeled HRP was applied
and incubated for 10 min, Opal 520 reagent was applied
and incubated for 10 min. The sections were counter-stainedwith DAPI and mounted in Vectorshield mounting medium(Vector Laboratories). Fluorescence images were captured
using a fluorescence microscope (BZ9000; Keyence, Osaka,
Japan).

Statistical analysis
The statistical significance of the differences between two
groups was determined using Student’s t test and Fisher’s
exact test. p values < 0.05 were considered significant.

Results
Patient characteristics
IDH-mutant and IDH-wildtype diffuse astrocytomas, IDHmutant and IDH-wildtype anaplastic astrocytomas, oligodendrogliomas, and anaplastic oligodendrogliomas (IDHmutant, 1p19q codel) accounted for 16, 5, 5, 9, 30, and 13
cases, respectively (Table 2). The proportion of cases with/without 1p19q codel or with/without ATRX mutation in thisstudy was similar to that in the 2 large cohorts (see also
Table 1). Progression-free survival and overall survival
times were 2.6 and 3.7 years, respectively. Before sampling, 21 patients (27%) received either or both chemo- andradiotherapy.

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Brain Tumor Pathology (2018) 35:106–113
Table 2  Patients’ characteristics
The number of cases
PFS (years)
OS (years)
GenderFemaleMale
Age (years, mean)(Range)WHO classification
Diffuse astrocytoma, IDH-mutant
Diffuse astrocytoma, IDH-wildtype
Anaplastic astrocytoma, IDH-mutant
Anaplastic astrocytoma, IDH-wildtype
Oligodendroglioma, IDH-mutant and 1p/19q codeleted
Anaplastic oligodendroglioma, IDH-mutant and1p/19q codeleted
Pretreatment
None
Radiotherapy
Chemotherapy
Chemo-radiotherapy

78
2.6
3.7
34
44
41.1
(17.6–72.9)
16
5
5
9
30
13

60
13
1
4

PFS progression-free survival, OS overall survival, WHO World
Healthcare Organization
Table 3  Correlation between loss of ATRX expression and ATRX
mutations

IHC-positive
IHC-negative

ATRX-wildtype

ATRX-mutant

Total

52
4
56

8
14
22

60
18
78
p < 0.00001

IHC immunohistochemistry

Correlation between loss of ATRX expression
and ATRX mutations
We performed IHC using 2 anti-ATRX antibodies with different epitopes. The results were consistent in all samples.
Of the 78 cases, 60 showed ATRX expression (IHCpositive) and 18 showed loss of ATRX expression (IHCnegative) in tumor cells (Table 3). Among the 60 IHCpositive cases, 52 (86.7%) were ATRX-wildtype and 8(13.3%) were ATRX-mutant by WES. Among the 18
IHC-negative cases, 14 (77.8%) were ATRX-mutant and 4(22.2%) were ATRX-wildtype by WES. All ATRX-mutant
tumors did not exhibit specific mutation patterns suchas splicing, frameshifts, or single-nucleotide variants by
WES (Table 4).

Brain Tumor Pathology (2018) 35:106–113

109

Table 4  ATRX mutation patterns by whole-exome sequence

Number of cases
Mutation pattern
Splicing
Frameshift insertion
Frameshift deletion
Nonsynonymous SNV
Stopgain SNV
Number of cases
Mutation pattern
Missense
Indel + nonsense + splice site

IHC-negative

IHC-positive

Total

14

8

22

4
1
4
2
3

0
2
1
3
2

4
3
5
5
5

IHC-negative

IHC-positive

Total

14

8

22

2
12

3
5

5
17
p = 0.3089

Does ATRX loss in immunohistochemical analysis
serve as a surrogate for 1p19q codeletion?

Case
No

0.2536
0.52730.613
0.30891

We next evaluated whether ATRX expression in IHC
serves as a surrogate for 1p19q codel. A spreadsheet ofthe results of IDH status, 1p19q codel status, ATRX-IHC,
and ATRX sequencing in 78 cases is shown in Fig. 1. Of
total, 11 cases with IDH mutation (Cases 41–51 in Fig. 1)
showed discrepancy between IHC-based ATRX expression and actual 1p19q status. Cases #41 and #42, forinstance, did not show ATRX expression in tumor cells,
while normal endothelial cells and normal brain cells
expressed ATRX. These tumors were initially diagnosed
as astrocytoma; however, genetic investigation of 1p19q
and ATRX revealed that they harbored 1p19q codel andwild-type ATRX, and thus the final diagnosis was oligodendroglioma, IDH-mutant, and 1p19q codel (Fig. 2a, b).

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78

First, as previously reported, ATRX mutation was mutually
exclusive with 1p19q codel (Table 1). Most IDH-mutant
LGGs with ATRX mutation did not have 1p19q codel (84,94, and 98% in this study, the JPN cohort, and the TCGA
cohort, respectively), and 93–98% of IDH-mutant LGGs
with 1p19q codel harbored ATRX-wildtype. In contrast,
52–71% of ATRX-wildtype tumors displayed 1p19q codel(Table 1). Second, a tumor with ATRX mutation did notalways exhibit loss of ATRX expression, as describedabove (Table 3).

p value

IDH

1p19q
ATRXIHC
ATRXsequence
IDH-mutant

1p19q co-del

ATRX-IHC loss

ATRX-sequence mutant

Fig. 1  Spreadsheet of IDH status, 1p19q codel, ATRX-IHC, and ATRX sequencing in each case. For the cases indicated in red, the pictures of
their pathology are shown in Figs. 2 and 3

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Brain Tumor Pathology (2018) 35:106–113

a

b

100µm
Case #41. Oligodendroglioma
IDH-mutant & 1p19q codeleted, Grade II
ATRX-wildtype (sequencing)

100µm
Case #42. Anaplasc Oligodendroglioma
IDH-mutant & 1p19q codeleted, Grade III
ATRX-wildtype (sequencing)

c

DAPI

50µm

ATRX

50µm

IDH-R132H

50µm

merge

50µm

Case #42. Anaplasc Oligodendroglioma
IDH-mutant & 1p19q codeleted, Grade III, ATRX-wildtype (sequencing)Fig. 2  Cases #41 (a Oligodendroglioma, IDH-mutant, 1p19q codeleted, Grade II) and #42 (b Anaplastic Oligodendroglioma, IDHmutant, 1p19q codeleted, Grade III) did not show ATRX expression
in tumor cells, while normal endothelial cells and normal brain cells
expressed ATRX. The diagnosis of these tumors was initially astrocytoma; however, following genetic investigation of 1p19q and ATRX,

they were found to harbor 1p19q codel and wildtype ATRX. Thus, thefinal diagnosis was oligodendroglioma, IDH-mutant, 1p19q codel. c
In Case #42, the multicolored co-immunofluorescent histology with
anti-ATRX and IDH1-R132H antibodies revealed that abundant
IDH-R132H-positive tumor cells did not merge with ATRX-positive
cells

Additionally, multicolored co-immunofluorescent histology with anti-ATRX and IDH1-R132H antibodies confirmed that abundant IDH1-R132H-positive tumor cells
did not merge with ATRX-positive cells (Fig. 2c). Cases#49 and #51 were ARTX-IHC positive, and thus based on
pathological diagnosis were oligodendroglioma (Fig. 3a,b). However, as tumor DNA did not display 1p19q codel,
the diagnosis changed to astrocytoma, IDH-mutant. Coimmunofluorescent histology showed co-expression of
ATRX and IDH1-R132H (Fig. 3c).
If our cohort had only been diagnosed based on IDH
status and IHC-based ATRX expression, 78 tumors
would have been subtyped as 48 oligodendroglial tumors,
16 IDH-mutant astrocytic tumors, and 14 IDH-wildtype
astrocytic tumors (Fig. 4). However, when the 1p19q codel
test was performed following ATRX-IHC, 8 of 48 ATRXIHC-positive tumors were classified as “1p19q non-codel”
and 3 of 16 ATRX-IHC-negative tumors were classified as“1p19q codel”; a total of 11 tumors (14%) were incorrectlyclassified (Fig. 4).

Discussion

13

The current study suggests that ATRX-IHC does nothave reliable sensitivity as a surrogate for 1p19q codel.
We found that diagnostic accuracy was 82.8% in 64 IDHmutant LGGs when only ATRX-IHC was used to examine
1p19q codel.
Among the 48 tumors showing positive ATRX, 8(16.6%) were found to be 1p19q non-codel. Similarly,
3 (18.7%) of 16 tumors with negative ATRX had 1p19q
codel. Thus, 11 of 78 tumors required reclassification.
We demonstrated that ATRX mutation was not wellcorrelated with the loss of expression in IHC. In contrast,
intratumor heterogeneity and sampling should be considered. Purkait et al. reported that ATRX-IHC was matchedto ATRX sequencing in only 50% of cases (3 of 6) [30].
They performed ATRX sequencing of microdissected sections showing a mosaic or negative pattern for ATRXIHC. Their results revealed that both mutational andBrain Tumor Pathology (2018) 35:106–113

111

a

b

100µm
Case #49. Diffuse Astrocytoma
IDH-mutant, Grade II

100µm
Case #51. Anaplasc Astrocytoma
IDH-mutant, Grade III

c

DAPI

50µm

ATRX

50µm

IDH-R132H

50µm

50µm

merge

Case #51. Anaplasc Astrocytoma
IDH-mutant, Grade III
Fig. 3  Cases #49 (a Diffuse astrocytoma, IDH-mutant, Grade II) and#51 (b Anaplastic astrocytoma, IDH-mutant, Grade III) are ATRXIHC-positive, which led to the pathological diagnosis of oligodendroglioma. However, as the tumor DNA did not display 1p19q codel,

Fig. 4  Flowchart of LGG
diagnosis in our cohort. If ourcohort had only been diagnosed based on IDH status andIHC-based ATRX expression,
78 tumors would have been
subtyped as 48 oligodendroglial tumors, 16 IDH-mutant
astrocytic tumors, and 14 IDHwild-type astrocytic tumors.
However, when the 1p19q codel
test was performed following
ATRX-IHC, 8 of 48 ATRXIHC-positive tumors wereclassified as “1p19q non-codel”
and 3 of 16 ATRX-IHCnegative tumors were classified
as “1p19q codel”; a total of 11
tumors (14%) were incorrectlyclassified

Layer1
IDH status

Layer2
ATRX-IHC64

IDH1-R132H
IHC posi ve,mutant by
sequencing

the diagnosis was changed to astrocytoma, IDH-mutant. c In Case
#51,co-immunofluorescent histology showed co-expression of ATRX
and IDH1-R132H

Final
diagnosis
48

Oligodendroglioma or
Anaplas c Oligodendroglioma
IDH-mutant and 1p19q co-deleted,
WHO grade II or grade III

Layer3
1p19q status

ATRX
IHC-positive

1p19q co-del

40

48
1p19q non-co-del

8Allsamples
1p19q co-del
IDH1-R132H
IHC nega ve,
wildtype by
sequencing

ATRX
IHC-negative

16

16

Layers 1+2+3;
43

Diffuse Astrocytoma or
Anaplas c Astrocytoma
IDH-mutant, WHO grade II or grade III

3

1p19q non-co-delLayers 1+2;
48

13
Layers 1+2;
16

Layers 1+2+3;
21

Diffuse Astrocytoma or
Anaplas c Astrocytoma
IDH-wildtype, WHO grade II or grade III

14

14

13

112

expressional statuses showed diverse patterns when sampling was performed from different sites, even within thesame tumor. Such intratumor heterogeneity may influence
the discrepancy between the results using ATRX-IHC and
WES. In contrast, non-recurrent ATRX mutation in glioma
may not lead to the loss of ATRX expression. Our study
showed that only splicing of ATRX resulted in IHC negativity, but frameshifts and single-nucleotide variants did
not affect the results of ATRX-IHC (Table 4). Although
we used two different antibodies for distinct epitopes inthis study, they may not be universally applicable for suchdifferent mutation patterns.
Whether ATRX-IHC can be used as a surrogate of
1p19q codel remains unclear. Ikemura et al. demonstrated that ATRX-IHC was 100% concordant with ATRX
sequencing in 19 LGGs as well as 1p19q status in 53 LGGs
[31]. In contrast, Purkait et al. and Reuss et al., respectively, reported that among 30 and 72 tumors with 1p19q
codel, 3 (10%) and 2 (3%) did not show ATRX expression[19, 30]. In European Association for Neuro-Ongology
guideline mentioned that ATRX-IHC might facilitate diagnosis but is not a substitute for 1p19q codel testing [32].
On the other hand, cIMPACT-NOW update2 suggests that
1p19q testing can be skipped in cases showing ATRX-IHC
negativity [33]. However, in our cohort, 3 (19%) of 16
IDH-mutant and ATRX-negative tumors harbored 1p/19q
codel, suggesting that the utility of ATRX-IHC as a surrogate of 1p19q codel may be still debatable.
In summary, we observed a dissociation between
ATRX-IHC positivity and the presence of actual 1p19q
codel in 11 of 64 IDH-mutant LGGs. In describing thecomplex IHC expression of ATRX somatic mutations, ourresults indicate the need for caution when using ATRXIHC as a surrogate of 1p19q status.
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) and Platform Project for Supporting Drug Discovery andLife Science Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED under Grant no.
JP17am0101078 (Y.K.). The authors thank Drs. Ichiro Ito and Reiko
Watanabe for their second pathological review.

References
1. Suzuki H, Aoki K, Chiba K et al (2015) Mutational landscapeand clonal architecture in grade II and III gliomas. Nat Genet
47:458–468
2. Ostrom QT, Gittleman H, Liao P et al (2017) CBTRUS Statistical Report: primary brain and other central nervous system
tumors diagnosed in the United States in 2010–2014. Neuro
Oncol 19:v1–v88

13

Brain Tumor Pathology (2018) 35:106–113
3. 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
4. Sanson M, Marie Y, Paris S et al (2009) Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker
in gliomas. J Clin Oncol 27:4150–4154
5. 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
6. van den Bent MJ, Brandes AA, Taphoorn MJ et al (2013) Adjuvant procarbazine, lomustine, and vincristine chemotherapy
in newly diagnosed anaplastic oligodendroglioma: long-term
follow-up of EORTC brain tumor group study 26951. J Clin
Oncol 31:344–350
7. 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
8. Cancer Genome Atlas Research N, Brat DJ, Verhaak RG et al(2015) Comprehensive, integrative genomic analysis of diffuse
lower-grade gliomas. N Engl J Med 372:2481–2498
9. Chaturbedi A, Yu L, Linskey ME et al (2012) Detection of
1p19q deletion by real-time comparative quantitative PCR.
Biomark Insights 7:9–17
10. Pinkham MB, Telford N, Whitfield GA et al (2015) FISHing
Tips: what every clinician should know about 1p19q analysis
in gliomas using fluorescence in situ hybridisation. Clin Oncol(R Coll Radiol) 27:445–453
11. Hayashi S, Sasaki H, Kimura T et al (2015) Molecular-genetic
and clinical characteristics of gliomas with astrocytic appearance and total 1p19q loss in a single institutional consecutive
cohort. Oncotarget 6:15871–15881
12. Jeuken J, Cornelissen S, Boots-Sprenger S et al (2006) Multiplex ligation-dependent probe amplification: a diagnostic tool
for simultaneous identification of different genetic markers inglial tumors. J Mol Diagn 8:433–443
13. Koschmann C, Calinescu AA, Nunez FJ et al (2016) ATRX loss
promotes tumor growth and impairs nonhomologous end joining
DNA repair in glioma. Sci Transl Med 8:328ra328
14. Nandakumar P, Mansouri A, Das S (2017) The role of ATRX
in glioma biology. Front Oncol 7:236
15. Schwartzentruber J, Korshunov A, Liu XY et al (2012) Driver
mutations in histone H3.3 and chromatin remodelling genes inpaediatric glioblastoma. Nature 482:226–231
16. Kannan K, Inagaki A, Silber J et al (2012) Whole-exome
sequencing identifies ATRX mutation as a key molecular determinant in lower-grade glioma. Oncotarget 3:1194–1203
17. Heaphy CM, de Wilde RF, Jiao Y et al (2011) Altered telomeresin tumors with ATRX and DAXX mutations. Science 333:425
18. 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
19. Reuss DE, Sahm F, Schrimpf D et al (2015) ATRX and IDH1R132H immunohistochemistry with subsequent copy number
analysis and IDH sequencing as a basis for an “integrated” diagnostic approach for adult astrocytoma, oligodendroglioma andglioblastoma. Acta Neuropathol 129:133–146
20. 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
21. Cai J, Zhang C, Zhang W et al (2016) ATRX, IDH1-R132H
and Ki-67 immunohistochemistry as a classification scheme forastrocytic tumors. Oncoscience 3:258–265

Brain Tumor Pathology (2018) 35:106–113
22. 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
23. Rogers TW, Toor G, Drummond K et al (2018) The 2016 revisionof the WHO classification of central nervous system tumours: retrospective application to a cohort of diffuse gliomas. J Neurooncol
137:181–189
24. Hewer E, Vajtai I, Dettmer MS et al (2016) Combined ATRX/
IDH1 immunohistochemistry predicts genotype of oligoastrocytomas. Histopathology 68:272–278
25. Wiestler B, Capper D, Holland-Letz T et al (2013) ATRX loss
refines the classification of anaplastic gliomas and identifies a
subgroup of IDH mutant astrocytic tumors with better prognosis.
Acta Neuropathol 126:443–451
26. Ceccarelli M, Barthel FP, Malta TM et al (2016) Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell 164:550–563
27. Aoki K, Nakamura H, Suzuki H et al (2018) Prognostic relevanceof genetic alterations in diffuse lower-grade gliomas. Neuro Oncol
20:66–77
28. Ogasawara S, Fujii Y, Kaneko MK et al (2016) Establishmentof anti-human ATRX monoclonal antibody AMab-6. Monoclon
Antib Immunodiagn Immunother 35:254–258

113
29. 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
30. Purkait S, Miller CA, Kumar A et al (2017) ATRX in diffuse gliomas with its mosaic/heterogeneous expression in a subset. Brain
Pathol 27:138–145
31. Ikemura M, Shibahara J, Mukasa A et al (2016) Utility of ATRX
immunohistochemistry in diagnosis of adult diffuse gliomas. Histopathology 69:260–267
32. Weller M, van den Bent M, Tonn JC et al (2017) European Association for Neuro-Oncology (EANO) guideline on the diagnosis
and treatment of adult astrocytic and oligodendroglial gliomas.
Lancet Oncol 18:e315–e329
33. 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. https​://doi.org/10.1007/s0040​
1-018-1826-y13