Biochemical and Biophysical Research Communications 433 (2013) 374–378

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Generation of a novel monoclonal antibody WMab-1 speciﬁc for
IDH2-R172W mutation
Yukinari Kato ⇑, Mika Kato Kaneko
Regional Innovation Strategy Support Program, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japana r t i c l e

i n f oArticle history:
Received 14 February 2013
Available online 20 March 2013
Keywords:
IDH2 mutation
R172W
Monoclonal antibody

a b s t r a c t
Isocitrate dehydrogenase 2 (IDH2) mutations have been detected in gliomas, cartilaginous tumors, andacute myeloid leukemias. IDH2 mutations are speciﬁc to a single codon in the conserved and functionally
important Arginine 172 (R172) or Arginine 140 (R140). To date, we have established speciﬁc monoclonal
antibodies (mAbs) against IDH2-R172K and IDH2-R172M. However, speciﬁc mAbs against IDH2-R172W
have not been reported. To establish IDH2-R172W-speciﬁc mAbs, we immunized rats with IDH2-R172W
peptides. Western-blot analysis showed that WMab-1 reacted with the IDH2-R172W recombinant
protein, not with IDH2-wild type (WT) or other IDH2 mutants, indicating that WMab-1 is IDH2R172W-speciﬁc. Furthermore, WMab-1 speciﬁcally stained the IDH2-R172W-expressing cells in immunocytochemistry, but did not stain IDH2-WT and IDH2-R172M-containing cells. WMab-1 also speciﬁcallystained the IDH2-R172W-expressing glioma cells in immunohistochemistry. This is the ﬁrst report toestablish an anti-IDH2-R172W-speciﬁc mAb, which could be useful in the diagnosis of IDH2-R172Wbearing tumors.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction
Isocitrate dehydrogenase 2 (IDH2) mutations have been detected in some gliomas [1–3], cartilaginous tumors [4–6], andacute myeloid leukemias (AML) [7,8]. IDH2 mutations have been
identiﬁed as early genetic alterations, and might be the initiating
event in these tumors [1–3]. The IDH2 mutations are remarkably
speciﬁc to a single codon in the conserved and functionally important Arginine 172 residue (R172) in gliomas. Of interest, IDH2
mutations of AML were discovered subsequently in Arginine 140
residue (R140), which are more frequent than R172 [7,8]. IDH2
mutations were found to possess the ability to change a-ketoglutarate to onco-metabolite R()-2-hydroxyglutarate (2-HG) [9].
To date, several monoclonal antibodies (mAbs) against IDH1/2
mutations have been reported [10–15]. Of those antibodies, weestablished HMab-1: an anti-IDH1-R132H-speciﬁc mAb [10],SMab-1: an anti-IDH1-R132S-speciﬁc mAb [11], GMab-m1: ananti-IDH1-R132G-speciﬁc mAb [15], KMab-1: an anti-IDH2R172K-speciﬁc mAb [14], and MMab-1: an anti-IDH2-R172M-speciﬁc mAb [14]. In our previous study, IDH1 mutation as evaluatedusing immunohistochemistry might be of greater prognostic signiﬁcance than histological grading alone in gliomas [10,16–19].

Abbreviations: IDH2, isocitrate dehydrogenase 2; mAb, monoclonal antibody.
⇑ Corresponding author. Fax: +81 22 717 8207.
E-mail addresses: yukinari-k@bea.hi-ho.ne.jp, yukinarikato@med.tohoku.ac.jp(Y. Kato).
0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2013.02.106

Here, we report a novel anti-IDH2-R172W-speciﬁc monoclonal
antibody WMab-1, which is expected to be extremely useful for
diagnosis of mutation-bearing gliomas.
2. Materials and methods
2.1. Cell lines and tissues
Sp2/0 and Chinese hamster ovary (CHO) cells were obtained
from the American Type Culture Collection (ATCC, Manassas, VA).
These cell lines were cultured at 37 °C in a humidiﬁed atmosphereof 5% CO2 and 95% air in RPMI 1640 medium (Wako Pure Chemical
Industries Ltd., Osaka, Japan), including 2 mM L-glutamine (Wako
Pure Chemical Industries Ltd.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies Corp., Carlsbad,
CA). One glioma patient (oligodendroglioma, WHO grade II–III),
who underwent primary surgery at Nagoya University Hospitaland one glioma patient (glioblastoma, WHO grade IV), who underwent primary surgery at Tsukuba University Hospital, were included in this study. Informed consent was obtained from eachpatient or the patient’s guardian for obtaining samples and for subsequent data analysis.
2.2. Hybridoma production
WMab-1 against IDH2-R172W was produced using rat medial
iliac lymph node methods [14,15]. Brieﬂy, WKY/Izm rats (JapanY. Kato, M.K. Kaneko / Biochemical and Biophysical Research Communications 433 (2013) 374–378

SLC Inc., Shizuoka, Japan) were immunized by injecting 170 lg ofsynthetic peptides of CGGTKPITIGWHAHGDQYKA, conjugated with
KLH together with Freund’s complete adjuvant (FCA; Sigma–Aldrich, St. Louis, MO) into footpad. The lymphocytes were fusedwith mouse myeloma Sp2/0 cells using polyethylene glycol (PEG)
methods. The culture supernatants were screened using ELISA forbinding to IDH2-R172W and IDH2-WT peptides, conjugated with
BSA.
2.3. Enzyme-linked immunosorbent assay (ELISA)
Synthetic peptides were immobilized, respectively, on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientiﬁc Inc.,
Waltham, MA) at 1 lg/ml for 30 min, respectively. After blocking
with SuperBlock T20 (PBS) Blocking Buffer in PBS (Thermo Fisher
Scientiﬁc Inc.), the plates were incubated with culture supernatant,followed by 1:1000 diluted peroxidase-conjugated anti-rat IgG(Dako, Glostrup, Denmark). The enzymatic reaction was conductedwith 1-Step Ultra TMB-ELISA (Thermo Fisher Scientiﬁc Inc.). The
optical density was measured at 655 nm with an iMark microplate
reader (Bio-Rad Laboratories Inc., Philadelphia, PA). These reactions were performed with a volume of 50 ll at 37 °C.
2.4. Plasmid preparation
Human IDH2 cDNA (Accession No. NM_002168) encoding a fulllength open reading frame (ORF) was obtained by PCR using a cDNA
derived from the U373 glioblastoma cell line [20] as a template. The
primer set for IDH2 was as follows: EcoRI-IDH2.F1, 50 -ccGAATTCgggATGGCCGGCTACCTGCGGG-30 and SalI-IDH2wterR1359, 50 gccgtcgacCTACTGCCTGCCCAGGGCTCT-30 . The ampliﬁed cDNA was
subcloned into a pcDNA3.1/V5-His-TOPO vector (Life Technologies
Corp.). Substitution of the Arginine 172 (R172) to appropriate amino acid codons (Lysine, Methionine, or Tryptophan) in IDH2 cDNAs
was accomplished using the QuikChange Lightning site-directedmutagenesis kit (Agilent Technologies, Santa Clara, CA). The fulllength IDH2-WT, IDH2-R172K, IDH2-R172M, and IDH2-R172W
were subcloned into an expression vector, pMAL-c2 (New England
Biolabs Inc., Beverly, MA), via EcoRI and SalI restriction sites. The
full-length IDH2 and each mutated ORF were ampliﬁed using theprimer set: EcoRI-IDH2.F1 and IDH2woterR1356-XhoI, 50 -taaCTCGAGcgCTGCCTGCCCAGGGCTCTG-30 . These PCR products were digested with EcoRI and XhoI restriction enzymes, and subcloned
into pcDNA3 vector (Life Technologies Corp.) together with thenucleotide sequence (ctcgagTGGCGTTGCCATGCCAGGTGCCGAAGATGATGTGGTGTAAtctaga), which encodes 12 amino acids,
GVAMPGAEDDVV (PA tag) [14].

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according to the manufacturer’s instructions. Stable transfectants
were selected by cultivating the transfectants in medium containing 1 mg/ml of G418 (Wako Pure Chemical Industries Ltd.). The
expression level of IDH2 was conﬁrmed using Western-blot
analysis.
2.6. Western-blot analyses
Cultured cell pellets were lysed with PBS with 1% TritonX-100
for 30 min on ice. The lysate supernatants were centrifuged for
15 min at 15,000 rpm to remove cellular debris. Cell lysates containing 10 lg of total protein were prepared for Western-blot analysis by boiling in SDS sample buffer (Nakalai Tesque, Inc., Kyoto,
Japan). They were electrophoresed on 5–20% polyacrylamide gels
(Wako Pure Chemical Industries Ltd.). The separated proteins weretransferred to a PVDF membrane (Bio-Rad Laboratories Inc.). After
blocking with 4% skim milk in PBS for 15 min, the membrane wasincubated with WMab-1 (1 lg/ml), 5F11 (anti-IDH2; 1 lg/ml;
Sigma–Aldrich), TMab-2 (anti-MBP tag [14]; 1 lg/ml), or NZ-1
(anti-PA tag [14]; 1 lg/ml) for 15 min; then with peroxidase-conjugated secondary antibodies (Dako, 1/1000 diluted) for 15 min,and developed with ECL-plus reagents (GE Healthcare, Buckinghamshire, UK) using SAYACA-IMAGER (DRC Co., Ltd., Tokyo, Japan).
2.7. Immunocytochemical (ICC) analyses
Cultured cells were ﬁxed with 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 20 min at room temperature, andpermeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells werethen treated with 10% normal goat serum in PBS (NGS/PBS) to
block nonspeciﬁc binding sites, and were incubated with 5 lg/ml
of WMab-1 containing 0.1% Triton X-100 overnight at 4 °C in amoist chamber. They were incubated with goat anti-rat IgG-Alexa
488 (dilution 1:400; Molecular Probes Inc., Eugene, OR) in PBS
containing 0.1% Triton X-100 for 1 h at room temperature. Cells
were also treated with TO-PRO-3 (Molecular Probes) to stain the

2.5. Protein expression using bacteria cells and mammalian cells
Competent Escherichia coli TOP-10 cells (Life Technologies
Corp.) were transformed with the plasmid, pMAL-IDH2-wild type(WT), pMAL-IDH2-R172K, pMAL-IDH2-R172M, and pMAL-IDH2R172W. Then, they were cultured overnight at 37 °C in LB medium(Life Technologies Corp.) containing 100 lg/ml ampicillin (Sigma–
Aldrich). Cell pellets were resuspended in phosphate bufferedsaline (PBS) with 1% Triton X-100 with 50 lg/ml aprotinin (Sigma–Aldrich). After sonication, the crude extracts were collectedby centrifugation (9000g 30 min, 4 °C). The supernatants were
loaded onto Amylose resin (New England Biolabs Inc.). The loaded
resins were washed extensively with column buffer (20 mM Tris–
HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA), and the fusion proteins
were eluted by column buffer with 10 mM maltose. CHO cells weretransfected with appropriate amounts of plasmids, pcDNA3/IDH2WT, pcDNA3/IDH2-R172K, pcDNA3/IDH2-R172M, or pcDNA3/
IDH2-R172W using Lipofectamine LTX (Life Technologies Corp.)

Fig. 1. Western-blot analyses by anti-IDH2 mAbs against MBP-fusion proteins.
Puriﬁed proteins (0.05 lg/lane) of E. coli-expressing IDH2 wild type (lane 1) andIDH2 mutants (lane 2, IDH2-R172K; lane 3, IDH2-R172M; lane 4, IDH2-R172W)
were electrophoresed under reducing conditions using 5–20% gels, and wereWestern-blotted with WMab-1, 5F11, and anti-MBP tag.

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Y. Kato, M.K. Kaneko / Biochemical and Biophysical Research Communications 433 (2013) 374–378

3. Results and discussion

to a-ketoglutarate in mitochondria [21]. To date, almost all IDH2
mutations have been identiﬁed at the critical isocitrate binding site
R172 codon or R140 codon in some malignant gliomas [1], cartilaginous tumors [4,5], and in acute myeloid leukemias [7]. The vast
majority of changes of IDH2 are heterozygous in the same way of
IDH1 mutation [1,2,22,23]. IDH1 mutations include R132H (664/716: 92.7%), R132C (29/716: 4.2%), R132S (11/716: 1.5%), R132G(10/716: 1.4%), and R132L (2/716: 0.2%); therefore, combinationof our established mAbs: an anti-R132H mAb (HMab-1) [10], ananti-R132S mAb (SMab-1) [11], and an anti-R132G mAb (GMabm1) [15] covers 95.6% of IDH1 mutations. It was also reported that
IDH2 mutations include R172K (20/31: 64.5%), R172M (6/31:
19.3%), and R172W (5/31: 16.2%) in gliomas [3]. Recently, we haveestablished KMab-1: anti-IDH2-R172K-speciﬁc mAb and MMab-1:
anti-IDH2-R172M-speciﬁc mAb [14]. Combination of our established anti-IDH2 mAbs could cover 83.8% of IDH2 mutations.
However, anti-IDH2-R172W mAbs have not been established. For
this study, we immunized rats with synthetic peptides of IDH2R172W. After cell fusion, the wells of hybridomas, which produced
IDH2-R172W-reactive/IDH2-wild type (WT)-nonreactive antibodies, were screened in ELISA. After limiting dilution, clone WMab1 (rat IgG2b, kappa) against IDH2-R172W was established.

3.1. Production of an IDH2-R172W-speciﬁc antibody

3.2. Speciﬁcity of an anti-IDH2-R172W mAb against IDH2 Mutants

The IDH2 gene at 15q26.1 encodes isocitrate dehydrogenase 2(IDH2), which catalyzes the oxidative carboxylation of isocitrate

To determine the speciﬁcity of WMab-1, the reactivity against
IDH2-WT and IDH2 mutants (MBP-IDH2-R172K, MBP-IDH2-cell nuclei. They were examined using confocal laser-scanningmicroscopy (LSM5 PASCAL; Carl Zeiss Inc., Jena, Germany). Then
ﬂuorescent images were processed using image-processing
software (Adobe Photoshop; Adobe Systems Inc.).
2.8. Immunohistochemical (IHC) analysis
IDH2-R172W protein expression was determined immunohistochemically in parafﬁn-embedded tumor specimens [14]. Brieﬂy,
4-lm-thick histologic sections were deparafﬁnized in xylene andrehydrated. Then, they were autoclaved in citrate buffer (pH 6.0;
Dako) for 20 min. Sections were incubated with 5 lg/ml of
WMab-1 overnight at 4 °C. Biotin-conjugated secondary anti-rat
IgG (Dako) was incubated for 30 min at room temperature followed by the peroxidase-conjugated biotin-streptavidin complex(Vectastain ABC Kit; Vector Laboratories Inc., Burlingame, CA) for
30 min at room temperature. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (DAB; Dako) for 10 min, andcounterstained with hematoxylin.

Fig. 2. The speciﬁcity of an anti-mutated IDH2 mAb, WMab-1. (A) Western-blot analyses by anti-IDH2 mAbs against mutated IDH2-expressing CHO cells. Total cell lysate
from CHO cells expressing IDH2 wild type (lane 1) and IDH2 mutants (lane 2, IDH2-R172K; lane 3, IDH2-R172M; lane 4, IDH2-R172W) were electrophoresed under reducing
conditions using 5–20% gels, and were Western-blotted with WMab-1, 5F11, and anti-PA tag. (B) Immunocytochemical analysis by WMab-1 against mutated IDH2transfected CHO cells. CHO cells were transfected by mutated IDH2. Each cell was stained by WMab-1, followed by anti-rat IgG-Alexa 488 antibody (green). Cells were also
treated with TO-PRO-3 to stain the cell nuclei (blue). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)Y. Kato, M.K. Kaneko / Biochemical and Biophysical Research Communications 433 (2013) 374–378

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3.3. Immunocytochemical (ICC) analyses by WMab-1 against IDH2R172W-expressing cell lines
Newly established WMab-1 was conﬁrmed to be an IDH2R172W-speciﬁc antibody in Western-blot analyses; therefore, we
performed immunocytochemical (ICC) analyses using IDH2-WT-,
IDH2-R172M-, or IDH2-R172W-transfected CHO cells. As depicted
in Fig. 2B, WMab-1 reacted with only CHO/IDH2-R172W, not with
CHO/IDH2-wild type (WT) and CHO/IDH2-R172M. Immunostaining of WMab-1 for CHO/IDH2-R172W demonstrated cytosolic patterns. These results indicate that WMab-1 is useful for detecting
IDH2-R172W in ICC, suggesting that WMab-1 could be also usefulin immunohistochemical analyses for detection of IDH2-R172W.
3.4. Immunohistochemical (IHC) analysis by WMab-1 against IDH2R172W-bearing gliomas
We next performed immunohistochemistry (IHC) of WMab-1
against IDH2-R172W-positive gliomas. Typical results are shownin Fig. 3. WMab-1 stained almost all tumor cells of IDH2-R172Wpositive gliomas (Fig. 3A), although no staining was observed in
IDH1-WT gliomas (Fig. 3B). These results indicate that WMab-1
is useful in IHC analyses for detection of IDH2-R172W mutation.
In near future, combination of WMab-1 with previously established anti-mutated IDH1 mAbs (HMab-1 [10], SMab-1 [11],GMab-m1 [15], GMab-r1 [24], LMab-1 [24]) and anti-mutated
IDH2 mAbs (KMab-1 [14], MMab-1 [14]) should increase the diagnostic rate using IHC against glioma patients.
In conclusion, an established anti-IDH2-R172W-speciﬁc mAb
WMab-1 is expected to be extremely useful for diagnosis and biological evaluation of mutation-bearing gliomas in combination
with anti-mutated IDH1/2-speciﬁc monoclonal antibodies.
Additional IDH1/2 mutation-speciﬁc antibodies must be developed
for pathological diagnosis of gliomas, cartilaginous tumors, and
AML.
Fig. 3. Immunohistochemical analyses by WMab-1 against glioma tissues. Glioma
tissues possessing IDH2-R172W (A) or IDH2-WT (B) were stained with WMab-1.
Magniﬁcation: 400.

R172M, MBP-IDH2-R172W) was investigated using Western-blot
analyses. As shown in Fig. 1, WMab-1 recognized only MBPIDH2-R172W protein, not with the other proteins (MBP-IDH2WT, MBP-IDH2-R172K, MBP-IDH2-R172M), indicating that
WMab-1 speciﬁcally recognizes MBP-IDH2-R172W protein. In contrast, clone 5F11 recognized MBP-IDH2-WT and all MBP-IDH2mutant proteins. These results indicate that WMab-1 is useful indetecting IDH2-R172W speciﬁcally. To exclude the possibility thatthe speciﬁcity of WMab-1 was affected by MBP-tag, which wasused for the expression of IDH2 mutants in E. coli, another GSTtag was also used in the expression of IDH2 mutants: GST-IDH2-WT,
GST-IDH2-R172K, GST-IDH2-R172M, and GST-IDH2-R172W. Results show that WMab-1 recognized only GST-IDH2-R172W (datanot shown), suggesting that the reactivity of WMab-1 is observed
independently of those tag proteins.
We next performed Western-blot analyses against mutated
IDH2-expressing CHO cells. As shown in Fig. 2A, WMab-1 reactedwith only IDH2-R172W protein expressed in CHO cells, not withthe other proteins (IDH2-WT, IDH2-R172K, IDH2-R172M). In contrast, anti-IDH2 mAb (clone 5F11) strongly recognized only
IDH2-WT; weakly recognized mutated IDH2 proteins. 5F11 also
detected endogenous IDH2 expressed in CHO cells. These results
indicate that WMab-1 is useful in detecting IDH2-R172W expressed in mammalian cells.

Acknowledgments
We thank Yasukazu Hozumi and Kaoru Goto (Yamagata University) for their excellent advice about ICC, and thank Atsushi
Natsume, Fumiharu Ohka (Nagoya University), and Shingo Takano(University of Tsukuba) for providing us glioma specimen. We also
thank Yuta Tsujimoto, Shunpei Morita, Ryo Yanagiya, Kana Nasu,
Hiroko Sasaki, Sachiko Suzuki, and Junko Aita (Yamagata University) for their excellent technical assistance. This work was supported in part by Platform for Drug Discovery, Informatics, andStructural Life Science (M.K.K., Y.K.), by Regional Innovation Strategy Support Program (Y.K.), by KAKENHI (23790185, 23701043): a
Grant-in-Aid for Young Scientists (B) (M.K.K., Y.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan,
and by Intelligent Cosmos Academic Foundation (Y.K.).
References[1] H. Yan, D.W. Parsons, G. Jin, R. McLendon, B.A. Rasheed, W. Yuan, I. Kos, I.
Batinic-Haberle, S. Jones, G.J. Riggins, H. Friedman, A. Friedman, D. Reardon, J.
Herndon, K.W. Kinzler, V.E. Velculescu, B. Vogelstein, D.D. Bigner, IDH1 andIDH2 mutations in gliomas, N. Engl. J. Med. 360 (2009) 765–773.
[2] Y. Sonoda, T. Kumabe, T. Nakamura, R. Saito, M. Kanamori, Y. Yamashita, H.
Suzuki, T. Tominaga, Analysis of IDH1 and IDH2 mutations in Japanese glioma
patients, Cancer Sci. 100 (2009) 1996–1998.
[3] C. Hartmann, J. Meyer, J. Balss, D. Capper, W. Mueller, A. Christians, J. Felsberg,
M. Wolter, C. Mawrin, W. Wick, M. Weller, C. Herold-Mende, A. Unterberg, J.W.
Jeuken, P. Wesseling, G. Reifenberger, A. von Deimling, Type and frequency of
IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial
differentiation and age: a study of 1,010 diffuse gliomas, Acta Neuropathol.
118 (2009) 469–474.
[4] T.C. Pansuriya, R. van Eijk, P. d’Adamo, M.A. van Ruler, M.L. Kuijjer, J. Oosting,
A.M. Cleton-Jansen, J.G. van Oosterwijk, S.L. Verbeke, D. Meijer, T. van Wezel,
K.H. Nord, L. Sangiorgi, B. Toker, B. Liegl-Atzwanger, M. San-Julian, R. Sciot, N.

378

Y. Kato, M.K. Kaneko / Biochemical and Biophysical Research Communications 433 (2013) 374–378

Limaye, L.G. Kindblom, S. Daugaard, C. Godfraind, L.M. Boon, M. Vikkula, K.C.
Kurek, K. Szuhai, P.J. French, J.V. Bovee, Somatic mosaic IDH1 and IDH2
mutations are associated with enchondroma and spindle cell hemangioma in
Ollier disease and Maffucci syndrome, Nat. Genet. 43 (2011) 1256–1261.
[5] M.F. Amary, S. Damato, D. Halai, M. Eskandarpour, F. Berisha, F. Bonar, S.
McCarthy, V.R. Fantin, K.S. Straley, S. Lobo, W. Aston, C.L. Green, R.E. Gale, R.
Tirabosco, A. Futreal, P. Campbell, N. Presneau, A.M. Flanagan, Ollier disease
and Maffucci syndrome are caused by somatic mosaic mutations of IDH1 and
IDH2, Nat. Genet. 43 (2011) 1262–1265.
[6] M.F. Amary, K. Bacsi, F. Maggiani, S. Damato, D. Halai, F. Berisha, R. Pollock, P.
O’Donnell, A. Grigoriadis, T. Diss, M. Eskandarpour, N. Presneau, P.C.
Hogendoorn, A. Futreal, R. Tirabosco, A.M. Flanagan, IDH1 and IDH2
mutations are frequent events in central chondrosarcoma and central andperiosteal chondromas but not in other mesenchymal tumours, J. Pathol. 224(2011) 334–343.
[7] P.S. Ward, J. Patel, D.R. Wise, O. Abdel-Wahab, B.D. Bennett, H.A. Coller, J.R.
Cross, V.R. Fantin, C.V. Hedvat, A.E. Perl, J.D. Rabinowitz, M. Carroll, S.M. Su,
K.A. Sharp, R.L. Levine, C.B. Thompson, The common feature of leukemiaassociated IDH1 and IDH2 mutations is a neomorphic enzyme activity
converting alpha-ketoglutarate to 2-hydroxyglutarate, Cancer Cell 17 (2010)
225–234.
[8] S. Chotirat, W. Thongnoppakhun, O. Promsuwicha, C. Boonthimat, C.U.
Auewarakul, Molecular alterations of isocitrate dehydrogenase 1 and 2 (IDH1and IDH2) metabolic genes and additional genetic mutations in newly
diagnosed acute myeloid leukemia patients, J. Hematol. Oncol. 5 (2012) 5.
[9] S. Gross, R.A. Cairns, M.D. Minden, E.M. Driggers, M.A. Bittinger, H.G. Jang, M.
Sasaki, S. Jin, D.P. Schenkein, S.M. Su, L. Dang, V.R. Fantin, T.W. Mak, Cancerassociated metabolite 2-hydroxyglutarate accumulates in acute myelogenous
leukemia with isocitrate dehydrogenase 1 and 2 mutations, J. Exp. Med. 207(2010) 339–344.
[10] S. Takano, Y. Kato, T. Yamamoto, M.K. Kaneko, E. Ishikawa, Y. Tsujimoto, M.
Matsuda, K. Nakai, R. Yanagiya, S. Morita, K. Tsuboi, A. Matsumura,
Immunohistochemical detection of IDH1 mutation, p53, and internexin as
prognostic factors of glial tumors, J. Neurooncol. 108 (2012) 361–373.
[11] M.K. Kaneko, W. Tian, S. Takano, H. Suzuki, Y. Sawa, Y. Hozumi, K. Goto, K.
Yamazaki, C. Kitanaka, Y. Kato, Establishment of a novel monoclonal antibody
SMab-1 speciﬁc for IDH1-R132S mutation, Biochem. Biophys. Res. Commun.
406 (2011) 608–613.
[12] Y. Kato, G. Jin, C.T. Kuan, R.E. McLendon, H. Yan, D.D. Bigner, A monoclonal
antibody IMab-1 speciﬁcally recognizes IDH1R132H, the most common
glioma-derived mutation, Biochem. Biophys. Res. Commun. 390 (2009) 547–
551.
[13] D. Capper, H. Zentgraf, J. Balss, C. Hartmann, A. von Deimling, Monoclonal
antibody speciﬁc for IDH1 R132H mutation, Acta Neuropathol. 118 (2009)
599–601.

[14] M.K. Kaneko, S. Morita, Y. Tsujimoto, R. Yanagiya, K. Nasu, H. Sasaki, Y. Hozumi,
K. Goto, A. Natsume, M. Watanabe, T. Kumabe, S. Takano, Y. Kato,
Establishment of novel monoclonal antibodies KMab-1 and MMab-1 speciﬁc
for IDH2 mutations, Biochem. Biophys. Res. Commun. 432 (2013) 40–45.
[15] Y. Kato, A. Natsume, M.K. Kaneko, A novel monoclonal antibody GMab-m1
speciﬁcally recognizes IDH1-R132G mutation, Biochem. Biophys. Res.
Commun. 432 (2013) 564–567.
[16] S. Takano, W. Tian, M. Matsuda, T. Yamamoto, E. Ishikawa, M.K. Kaneko, K.
Yamazaki, Y. Kato, A. Matsumura, Detection of IDH1 mutation in humangliomas: comparison of immunohistochemistry and sequencing, Brain Tumor
Pathol. 28 (2011) 115–123.
[17] K. Motomura, A. Natsume, R. Watanabe, I. Ito, Y. Kato, H. Momota, R.
Nishikawa, K. Mishima, Y. Nakasu, T. Abe, H. Namba, Y. Nakazato, H. Tashiro, I.
Takeuchi, T. Mori, T. Wakabayashi, Immunohistochemical analysis-based
proteomic subclassiﬁcation of newly diagnosed glioblastomas, Cancer Sci.
103 (2012) 1871–1879.
[18] M.J. Kwon, S.T. Kim, D.S. Kong, D. Lee, S. Park, S.Y. Kang, J.Y. Song, D.H. Nam, Y.
Kato, Y.L. Choi, Y.L. Suh, Mutated IDH1 is a favorable prognostic factor for type2 gliomatosis cerebri, Brain Pathol. 22 (2012) 307–317.
[19] A. Yoshikawa, M. Nakada, T. Watanabe, Y. Hayashi, H. Sabit, Y. Kato, S. Suzuki,
A. Ooi, H. Sato, J.I. Hamada, Progressive adult primary glioblastoma in the
medulla oblongata with an unmethylated MGMT promoter and without an
IDH mutation, Brain Tumor Pathol. (2012), http://dx.doi.org/10.1007/s10014012-0118-9.
[20] Y. Kato, M.K. Kaneko, A. Kuno, N. Uchiyama, K. Amano, Y. Chiba, Y. Hasegawa, J.
Hirabayashi, H. Narimatsu, K. Mishima, M. Osawa, Inhibition of tumor cellinduced platelet aggregation using a novel anti-podoplanin antibody reactingwith its platelet-aggregation-stimulating domain, Biochem. Biophys. Res.
Commun. 349 (2006) 1301–1307.
[21] S. Zhao, Y. Lin, W. Xu, W. Jiang, Z. Zha, P. Wang, W. Yu, Z. Li, L. Gong, Y. Peng, J.
Ding, Q. Lei, K.L. Guan, Y. Xiong, Glioma-derived mutations in IDH1
dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha, Science
324 (2009) 261–265.
[22] D.W. Parsons, S. Jones, X. Zhang, J.C. Lin, R.J. Leary, P. Angenendt, P. Mankoo, H.
Carter, I.M. Siu, G.L. Gallia, A. Olivi, R. McLendon, B.A. Rasheed, S. Keir, T.
Nikolskaya, Y. Nikolsky, D.A. Busam, H. Tekleab, L.A. Diaz Jr., J. Hartigan, D.R.
Smith, R.L. Strausberg, S.K. Marie, S.M. Shinjo, H. Yan, G.J. Riggins, D.D. Bigner,
R. Karchin, N. Papadopoulos, G. Parmigiani, B. Vogelstein, V.E. Velculescu, K.W.
Kinzler, An integrated genomic analysis of human glioblastoma multiforme,
Science 321 (2008) 1807–1812.
[23] J. Balss, J. Meyer, W. Mueller, A. Korshunov, C. Hartmann, A. von Deimling,
Analysis of the IDH1 codon 132 mutation in brain tumors, Acta Neuropathol.
116 (2008) 597–602.
[24] M.K. Kaneko, Y. Tsujimoto, Y. Hozumi, K. Goto, Y. Kato, Novel monoclonal
antibodies GMab-r1 and LMab-1 speciﬁcally recognize IDH1-R132G and IDH1R132L mutations, Monoclon. Antib. Immunodiagn. Immunother., in press.