Pathology International 2016; 66: 578–582

doi:10.1111/pin.12459

Short Communication

Immunohistochemistry using monoclonal antibody MsMab-2 isuseful to detect IDH1 R132L in intrahepatic cholangiocarcinoma

Akimasa Hayashi,1† Kento Misumi,1† Junji Shibahara,1 Norihiro Kokudo,2 Yukinari Kato3 and Masashi Fukayama1
1

Department of Pathology, Graduate School of Medicine, 2Hepato-Biliary-Pancreatic Surgery Division, Department ofSurgery, Graduate School of Medicine, University of Tokyo, Tokyo and 3Department of Regional Innovation, Tohoku
University Graduate School of Medicine, Sendai, Miyagi, Japan

Immunohistochemical analysis using specific antibodies is a
useful and convenient method to detect proteins altered by somatic mutations. We previously generated the rat monoclonal
antibody MsMab-2, which recognizes isocitrate dehydrogenase (IDH)1 R132L and IDH2 R172M. In the present study, weused an immunohistochemical method to examine MsMab-2
immunoreactivity in 95 cases of intrahepatic cholangiocarcinoma, including five IDH1 R132L and one IDH2 R172M mutant
cases confirmed by direct sequencing. Tissue microarray section slides of all IDH1/2-mutant and wild-type cases, as well as
whole section slides of IDH1 R132L and IDH2 R172M cases
were immunostained using an autostainer. All IDH1 R132L
cases showed positive staining for MsMab-2, while other
IDH1/2 mutant and IDH1/2 wild-type cases were negative.
Tumor cells of the immunopositive cases invariably showedstrong reactivity using whole-section slides. We consider
immunohistochemical analysis using MsMab-2 to be a usefulmeans of detecting IDH1 R132L. Further analysis of its
effectiveness against IDH2 R172M is necessary because of
the small sample size in this study.
Key words: IDH,
immunohistochemistry,
cholangiocarcinoma, MsMab-2

intrahepatic

Cholangiocarcinoma is a malignant biliary neoplasm with agenerally poor patient prognosis, which appears to be increasing in overall incidence.1 Recent genome-wide studies haverevealed genomic alternations including somatic mutations ofgenes such as isocitrate dehydrogenases 1/2 (IDH1/2), KRAS,
BRCA associated protein-1 (BAP1), AT-rich interactive domain
1 A (ARID1A), and Polybromo 1 (PBRM1).2,3
In genes with mutation hot spots that dramatically alter thefunctioning of their encoded proteins, immunohistochemical

analysis with specific antibodies is useful to detect protein alterations. For example, the BRAF mutation status can be detected easily and precisely using a monoclonal antibody
against BRAF V600E.4 IDH1 and IDH2 are also good candidates because they each have a mutation hot spot, in IDH1
exon 4 at codon 132 and in IDH2 exon 4 at codon 172, asconfirmed in several previous studies2,3,5,6 and the Catalogueof Somatic Mutations in Cancer (COSMIC) (http://cancer.
sanger.ac.uk/cosmic). Out of four major mutations and resultant alterations in IDH1, previous studies succeeded in
immunohistochemically detecting IDH1 R132H,7 but none
could detect IDH1 R132L.
In the present study, we focused on IDH1 R132L and theusefulness of immunohistochemical analysis with the rat
monoclonal antibody MsMab-2 which recognizes IDH1
R132L and IDH2 R172M. We previously generated this antibody, and examined its reliability and specificity using western blotting and enzyme-linked immunosorbent assays,8 but
did not confirm this with an immunohistochemical assay using
formalin-fixed, paraffin-embedded (FFPE) samples. Although
IDH1 R132L has been identified in just 1 % of cholangiocarcinoma cases,9,10 our mutational analysis showed that it
was present in 5 % of cases (5/95) of intrahepatic cholangiocarcinoma (ICC). In this study we aimed not only to confirm
the sensitivity and specificity of MsMab-2 using FFPE surgicalsamples, but also to examine its usefulness in the routine
diagnosis of ICC.
MATERIALS AND METHODSPatients and tissue microarrays

Correspondence: Junji Shibahara, M.D. Department of Pathology,
Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo, 113-0033 Japan. Email: jshiba-tky@umin.ac.jp†These authors contributed equally to this work.
Received 26 April 2016. Accepted for publication 17 August 2016.
© 2016 Japanese Society of Pathology and John Wiley & Sons
Australia, Ltd

A total of 95 patients with primary ICC who underwent surgical treatment at The University of Tokyo Hospital fromJanuary 1, 1995 to December 31, 2013 were enrolled in thisstudy. Pathology reports and all tissue slides were reviewed
for all patients to confirm the diagnoses. Distal (extrahepatic)

IHC to detect IDH1 R132L in ICC

and perihilar cholangiocarcinomas and intraductal papillary
mucinous neoplasms were excluded.
Tissue microarrays (TMAs) were generated for all 95 ICC
cases according to well-established procedures. In brief, twotissue cores (2 mm diameter each) were punched out of eachdonor paraffin block and transferred to each of the recipient
TMA blocks.

579

IDH1/2 mutational status
The IDH1/2 mutational status was analyzed in our previous
study.11 In short, tumor DNA was extracted from FFPE tissue
blocks and amplified by PCR with paired primers focusing on
exon 4 at codon 132 of IDH1 and exon 4 at codon 172 of
IDH2. Amplified DNA was analyzed using direct sequencing.

Figure 1 Representative photomicrographs of immunohistochemistry analysis using MsMab-2 in five ICC cases with IDH R132L. Images of H&E
staining (a, d, g, j, and m) and immunohistochemical staining using MsMab-2 (b, e, h, k, and n), as well as results of sequencing analyses (c, f, i, l,and o), are shown for cases of ICC with IDH R132L. Each column shows data from the same case. Scale bar represents 100 μm.
© 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd

580

A. Hayashi et al.

IDH1 R132 mutations were observed in 19 of 95 (20.0 %) ICC
cases, including five with IDH1 R132L, 11 with IDH1 R132C,
and three with IDH1 R132G. IDH2 R172 mutations wereconfirmed in just two cases (2.1 %), including one with IDH2
R172M and one with IDH2 R172K.

were subjected to immunohistochemical staining using a
Ventana BenchMark XT automated immunostainer (Roche,
Basel, Switzerland). Whole tissue sections of representative
areas of tumors with IDH1 R132L and IDH2 R172M were
immunostained using the same protocols.

Immunohistochemistry Using the Monoclonal Antibody
MsMab-2

Ethics

The rat monoclonal antibody MsMab-2, shown to recognize
IDH1 R132L and IDH2 R172M in a previous Western
blotting analysis, was generated previously.8 TMA sections(3 μm-thick), including both IHD1/2 wild-type and mutant cases,

The University of Tokyo Medical Research Center Ethics Committee approved the study. Clinical samples were collectedfollowing written informed consent from patients under The
University of Tokyo Institutional guidelines for the study ofhuman tissues.

Figure 2 Representative photomicrographs of
immunohistochemistry analysis using MsMab-2
in ICC with IDH1 R132C, IDH1 R132G, IDH2
R172M, and wild-type IDH 1/2. Images of H&E
staining (a, c, e, and g) and immunohistochemical
staining using MsMab-2 (b, d, f, and h) are shownfor cases of ICC with IDH1 R132C (a, b), IDH1
R132G (c, d), IDH2 R172M (e, f), and wild-type
IDH 1/2 (g, h). Scale bar represents 100 μm.
© 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd

IHC to detect IDH1 R132L in ICC

RESULTS
Detection of IDH1 R132L Using MsMab-2
All IDH1 R132L mutant cases showed immunohistochemical
positivity for MsMab-2, while other IDH1 R132 mutant andwild-type cases had no immunoreactivity (Fig. 1, Fig. 2, andTable 1). Tumor cells invariably showed strong immunoreactivity to MsMab-2 in the analysis of whole tissue sections. IDH2
R172M and IDH2 R172K cases showed negative immunostaining for MsMab-2.

DISCUSSION
Our study demonstrated that IDH1 R132L could be precisely
identified using FFPE samples with the rat monoclonal antibody
MsMab-2. The number of antibodies specific to mutant IDH1/2
and available for FFPE specimens is limited7,12 and MsMab-2
was the first antibody to be developed that can detect IDH1
R132L specifically using tissue sections from FFPE samples.
We therefore consider immunohistochemical analysis to be auseful method of detecting IDH1 R132L and diagnosing ICC.
Though sequencing analysis is effective at detecting somatic
mutations, immunohistochemical analysis is more convenient,
especially for pathologists. Additionally, it is advantageouswhen PCR proves difficult because of high DNA fragmentation.
We previously reported MsMab-2 immunoreactivity against
IDH2 R172M using western blot analysis,8 but our present findings revealed no immunoreactivity in the immunohistochemical
analysis of an IDH2 R172M case using an FFPE sample.
While antibodies may differ in immunoreactivity between immunohistochemical and western blot analysis,13 we think thatfurther analyses are necessary to confirm MsMab-2 immunoreactivity because of the small number of IDH2 R172M cases inthis study.
An IDH mutation was first discovered in colorectal cancers
during the consensus coding sequence project.14 Since then,genome studies have identified somatic IDH mutations in several cancers, including glioma,5 leukemia,15 and intrahepaticcholangiocarcinoma.3 Most IDH1 mutations occur at arginine
132; this is thought to be a functional domain, so the mutation
presumably results in oncogenic enzymatic activity.16 Additionally, a molecular study showed that mutant IDH1 can prevent581

histone demethylation through 2-hydroxyglutarate production
from α-ketoglutarate.17 This epigenetic dysregulation and thefollowing expression profile alteration are considered topromote apoptosis resistance, migration, and invasion.18
Out of six IDH1 R132 variants, R132C, R132H, R132G,
R132L, and R132S have been identified in previous studies,
and R132C and R132H are the most common.19 IDH1 R132
mutant expression is known to vary by tumor type, but the clinicopathological features of each IDH1 R132 variant remains unclear. Regarding cholangiocarcinoma specifically, no studies
have identified differences in clinicopathological characteristics
between IDH1 R132 variants.19 Our previous study identified
no statistically significant differences in the characteristics of
R132C, R132G, and R132L, though the sample size was limited (data not shown).
IDH1 R132L is not a common IDH1 mutation according to theCatalogue of Somatic Mutations in Cancer (COSMIC) (http://
cancer.sanger.ac.uk/cosmic). However, a study of IDH mutations in mesenchymal tumors found that IDH1 R132L was morecommon in some types of tumors than expected.6 Moreover,
we previously showed11 that IDH1 R132L was more frequent
in ICC than in previous studies.3,9 Because ICC ratios in liver tumors differ between countries,20 this suggests that IDH1 R132L
frequencies in ICC differ between regions, races, or as yet
undetermined risk factors.
In conclusion, we demonstrate the precise immunohistochemical detection of IDH1 R132L in ICC FFPE samples using
the rat monoclonal antibody MsMab-2. We believe that thismethod will be useful in the detection or classification of a variety of tumors, including ICC, in routine diagnosis. In addition,
we anticipate that other antibodies specific for the IDH1 R132
mutant, which is more frequent in ICC (i.e., IDH1 R132C), will
be available in the future.
ACKNOWLEDGMENTS
This work was supported in part by the Practical Research for
Innovative Cancer Control from Japan Agency for Medical Research and development, AMED (Y.K.); by JSPS KAKENHI
Grant Number 16 K10748 (Y.K.), by the Platform for Drug
Discovery, Informatics, and Structural Life Science (PDIS) from
AMED (Y.K.).
DISCLOSURE STATEMENT

Table 1

Genetic IDH status based on MsMab-2 immunoreactivity

IDH status

No. of Positive Cases

Percentage

Wild type
IDH1-R132L
IDH1-R132C
IDH1-R132G
IDH2-R172M
IDH2-R172K

0/74
5/5
0/11
0/3
0/1
0/1

0%
100 %
0%
0%
0%
0%

None declared.
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© 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd