RAPID COMMUNICATION
`
`Human Mutation
`
`OFFICIAL JOURNAL
`
`IDH1 Mutations at Residue p.R132 (IDH1R132) Occur
`Frequently in High-Grade Gliomas But Not in Other Solid
`Tumors
`
`www.hgvs.org
`
`Fonnet E. Bleeker,1,2 Simona Lamba,2 Sieger Leenstra,3,4 Dirk Troost,5 Theo Hulsebos,6 W. Peter Vandertop,1,7
`Milo Frattini,8,9 Francesca Molinari,9 Margaret Knowles,10 Aniello Cerrato,11 Monica Rodolfo,8 Aldo Scarpa,12
`Lara Felicioni,13 Fiamma Buttitta,13 Sara Malatesta,13 Antonio Marchetti,13 and Alberto Bardelli2,14
`1Neurosurgical Center Amsterdam, Location Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
`2Laboratory of Molecular Genetics, The OncoGenomics Center, Institute for Cancer Research and Treatment, University of Torino, Medical
`School, Candiolo, Italy
`3Department of Neurosurgery, St. Elisabeth Ziekenhuis, Tilburg, The Netherlands
`4Department of Neurosurgery, Erasmus Medical Center, Rotterdam, The Netherlands
`5Departments of Neuropathology, The Netherlands
`6Department of Neurogenetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
`7Neurosurgical Center Amsterdam, Location VU University Medical Center, Amsterdam, The Netherlands
`8Department of Experimental Oncology, Istituto Nazionale Tumori; Milan, Italy
`9Laboratory of Molecular Diagnostic Institute of Pathology, Locarno, Switzerland
`10Section of Experimental Oncology, Leeds Institute for Molecular Medicine, Leeds, United Kingdom
`11Institute of Endocrinology and Experimental Oncology, National Council of Research, Naples, Italy
`12Department of Pathology, Section of Anatomic Pathology, University of Verona, Verona, Italy
`13Clinical Research Center, Center of Excellence on Aging, University-Foundation, Chieti, Italy
`14FIRC Institute of Molecular Oncology, Milan, Italy
`
`Communicated by Richard Wooster
`Received 1 October 2008; accepted revised manuscript 10 October 2008.
`Published online 31 December 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/humu.20937
`
`ABSTRACT: Systematic sequence profiling of the Glio-
`blastoma Multiforme (GBM) genome has recently led to
`the identification of somatic mutations in the isocitrate
`dehydrogenase 1 (IDH1) gene. Interestingly, only the
`evolutionarily conserved residue R132 located in the
`substrate binding site of IDH1 was found mutated in
`GBM. At present, the occurrence and the relevance of
`p.R132 (IDH1R132) variants in tumors other than GBMs
`is largely unknown. We searched for mutations at
`position R132 of the IDH1 gene in a panel of 672 tumor
`samples. These included high-grade glioma, gastrointest-
`inal stromal tumors (GIST), melanoma, bladder, breast,
`colorectal, lung, ovarian, pancreas, prostate, and thyroid
`carcinoma specimens. In addition, we assessed a panel of
`84 cell
`lines from different tumor lineages. Somatic
`mutations affecting the IDH1R132 residue were detected
`
`Additional Supporting Information may be found in the online version of this
`
`article.
`
`
`Correspondence to: Alberto Bardelli, Laboratory of Molecular Genetics, Institute
`
`for Cancer Research and Treatment, University of Torino; Medical School, Str prov
`
`142Km 3.95; Candiolo (TO), 10060, Italy. E-mail: a.bardelli@unito.it.
`
`Contract grant sponsor: The Italian Association for Cancer Research (AIRC; A.B.);
`
`Italian Ministry of Health, Regione Piemonte (A.B.); Italian Ministry of University and
`
`Research, CRT Progetto Alfieri (A.B.); Fondazione Monte dei Paschi di Siena, Siena,
`
`Italy (A.S.); Association for International Cancer Research (AICR-UK; A.B.) and EU
`
`FP6; Grant number: 037297 (A.B.).
`
`in 20% (23 of 113) high-grade glioma samples. In
`addition to the previously reported p.R132H and
`p.R132S alleles, we identified three novel
`somatic
`mutations (p.R132C, p.R132G, and p.R132L) affecting
`residue IDH1R132 in GBM. Strikingly, no IDH1 muta-
`tions were detected in the other tumor types. These data
`indicate that cancer mutations affecting IDH1R132 are
`tissue-specific, and suggest that it plays a unique role in
`the development of high-grade gliomas.
`Hum Mutat 30, 7–11, 2009.
`& 2008 Wiley-Liss, Inc.
`
`KEY WORDS: cancer; somatic mutation; IDH1; GBM;
`HGG
`
`Introduction
`
`tumor genomes is taking an
`The molecular profiling of
`enormous spurt these days. Genome-wide sequencing analyses
`have been performed in colorectal and breast cancer (Sjo¨blom
`et al., 2006; Wood et al., 2007), and most recently the same
`approach has been performed in pancreatic ductal adenocarcino-
`ma (PDAC) (Jones et al., 2008) and glioblastoma multiforme
`(GBM) (Parsons et al., 2008). These mutational efforts have led to
`the identification of novel somatic mutations in genes that had
`not been previously linked to tumorigenesis. Of particular interest
`
`& 2008 WILEY-LISS, INC.
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`screen revealed 12% somatic
`the GBM mutational
`that
`is
`mutations in the IDH1 gene (MIM# 147700) (Parsons et al.,
`2008). The IDH1 mutations were found predominantly in the
`group of secondary GBMs and younger patients. Furthermore, the
`patients with mutated IDH1 had a significantly longer survival
`(Parsons et al., 2008). Other tumor type datasets analyzed for this
`gene have been relatively small thus far, and the reported mutation
`frequencies are generally low (0 of 11 breast, 1 of 11 colon) (Wood
`et al., 2007) (http://www.sanger.ac.uk/cosmic).
`IDH1 encodes isocitrate dehydrogenase 1 (Geisbrecht and
`Gould, 1999), an enzyme that catalyzes the oxidative decarboxy-
`lation of isocitrate to a-ketoglutarate (Koshland et al., 1985). This
`reaction leads to NADPH production, and is thought to play a
`role in the cellular control of oxidative damage (Lee et al., 2002).
`IDH1 is
`localized within the cytoplasm and peroxisomes
`(Geisbrecht and Gould, 1999). Two different mutations in IDH1
`have been described in GBM, both affecting the amino acid
`arginine at position 132 and leading to amino acid residue
`substitutions (p.R132H and p.R132S). R132 is evolutionarily
`highly conserved, and is localized in the substrate binding site of
`IDH1, where hydrophilic interactions between R132 and both the
`a- and b-carboxylate of isocitrate are formed (Xu et al., 2004). The
`IDH1p:R132H and p:R132S changes might affect these interactions and
`its enzymatic activity (Parsons et al., 2008). In this study we have
`investigated the mutational status of IDH1 in 672 tumor samples
`and 84 cancer cell lines.
`
`Materials and Methods
`
`High-grade glioma (HGG; WHO grade III and grade IV) tumor
`samples (GBM, anaplastic astrocytoma, and anaplastic oligoden-
`droglioma) and the matched normal DNA samples were obtained
`from the tumor bank maintained by the Departments of
`
`the Academic Medical
`Neurosurgery and Neuropathology at
`Center (Amsterdam, The Netherlands). DNA of melanoma,
`colorectal cancer, and gastrointestinal stromal tumors (GIST)
`samples was obtained from the Department of Experimental
`Oncology at the Istituto Nazionale Tumouri (Milan, Italy). DNA
`of PDAC xenografts was obtained from the Department of
`Pathology, Section of Anatomic Pathology at the University of
`Verona (Verona, Italy). DNA of breast, lung, ovarian, and thyroid
`(papillary carcinoma) cancer samples was obtained from the
`Clinical Research Center, Center of Excellence on Aging at the
`University-Foundation (Chieti, Italy). Additional DNA samples of
`thyroid carcinomas (medullary histotype), extracted from frozen
`tissues, were obtained from the Department of Cellular Biology
`and Molecular Pathology at the University of Naples (Naples,
`Italy). DNA of bladder cancer samples was obtained from the
`Section of Experimental Oncology at the Leeds Institute for
`Molecular Medicine (Leeds, UK). Tumor databases are listed in
`Table 1, and have been previously validated by showing that
`somatic mutations in common cancer genes could be detected at
`the expected frequencies.
`In addition, a panel consisting of 84 cell lines from multiple
`tumor lineages was screened for IDH1R132
`mutations. Cell line
`details are shown in Supp. Table S1. The NCI-60 panel (the 60
`human cancer cell lines of the National Cancer Institute) was
`obtained from ATCC (Middlesex, UK). In addition, 16 astro-
`cytoma cell lines were included: the cell lines CCF-STTG1, Hs683,
`U87MG, U118MG, U251MG, U373MG, T98G (ATCC, Middlesex,
`UK), GAMG (Deutsche Sammlung von Mikroorganismen und
`Zellkulturen, Braunschweig, Germany), SKMG-3 (a gift of Dr. C.Y.
`Thomas, University of Virginia Division of Hematology/Oncol-
`ogy, Charlottesville, VA), D384MG, SF763 (gifts of Dr. M.L.
`Lamfers, Department of Neurosurgery, Free University, Amster-
`dam, The Netherlands), SF126 (a gift of Dr. C. Van Bree,
`
`Table 1.
`
`IDH1R132 mutations are specific for high grade gliomas
`
`Tumor type
`
`Histotype
`
`Number of samples analysed
`
`Number of mutated samples
`
`P-value (Fisher’s exact test)
`
`High grade glioma
`
`Bladder
`Breast
`
`Colorectal
`GIST
`Lung
`
`Melanoma
`Thyroid
`
`Ovary
`Pancreas
`Prostate
`
`Total
`GBM primary
`GBM secondary
`AA
`AO
`Transitional Cell
`Total
`Ductal
`Lobular
`Medullary
`Mucinous
`Adenocarcinoma
`
`Total
`Adenocarcinoma
`Carcinoid
`Small Cell
`
`Total
`Medullary
`Papillary
`Adenocarcinoma
`Ductal Adenocarcinoma
`
`113
`94
`15
`2
`2
`34
`127
`48
`45
`17
`17
`128
`25
`107
`84
`7
`16
`23
`42
`21
`21
`46
`23
`4
`
`23
`11
`11
`0
`1
`0
`0
`
`0
`0
`0
`
`0
`0
`
`0
`0
`
`0.002179
`8,03E-09
`0.000292
`0.000295
`0.041578
`0.041578
`7,26E-09
`0.014124
`8,20E-09
`0.000001
`0.343849
`0.073930
`0.013501
`0.000576
`0.023904
`0.023904
`0.000284
`0.013501
`0.584147
`
`Tumor samples, tumor type, histotype, the number of samples analysed and the number of mutated samples are indicated. In addition, P-values of the Fisher’s exact test, used
`to determine the tissue specificity for IDH1R132
`mutations in high grade gliomas, are listed. Abbreviations: AA; Anaplastic Astrocytoma, AO; Anaplastic Oligodendroglioma,
`GBM; Glioblastoma Multiforme, GIST; Gastrointestinal Stromal Tumors.
`
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`Table 2. Mutations affecting IDH1R132 identified in high grade
`gliomas
`
`IDH1 mutation
`
`Nucleotide
`
`Amino
`Acid
`
`Number of
`mutated samples
`
`Histology of
`mutated samples
`
`Previously
`described
`
`c.394C4T p.R132C
`c.394C4G p.R132G
`c.394C4A p.R132S
`c.395G4T p.R132L
`c.395G4A p.R132H
`
`3
`1
`1
`1
`17
`
`GBM
`GBM
`AO
`GBM
`GBM
`
`yes
`no
`yes
`no
`yes
`
`The nucleotide and amino acid changes are listed alongside the number and
`histology of the mutated samples. In addition, we indicate whether the mutation has
`been described before in high grade gliomas. The nucleotide numbering uses the A of
`the ATG translation initiation start site as nucleotide 11, based on reference sequence
`NM__005896.2. All mutations are heterozygous. Abbreviations: AO; Anaplastic
`Oligodendroglioma, GBM; Glioblastoma Multiforme.
`
`Cancer cell lines in which the IDH1R132 mutations
`Table 3.
`were analyzed
`
`Tumor type
`
`Astrocytoma
`Bladder
`Breast
`Cervix
`Colon
`HNSCC (tongue)
`Kidney
`Leukemia
`Lung
`Melanoma
`Mesothelioma
`Oesophageus
`Ovary
`Prostate
`Thyroid
`
`Number of cell lines analysed
`
`20
`4
`7
`1
`9
`1
`6
`5
`9
`8
`1
`2
`8
`2
`1
`
`Cell lines are listed according to the tumor lineage from which they were originated.
`Abbreviation: HNSCC; Head and neck squamous cell carcinoma.
`
`University of Amsterdam, Laboratory for Experimental Oncology
`and Radiation Biology, Amsterdam, The Netherlands), A58 and
`A60 (gifts of Dr. A. van Tilborg and Dr. P. De Witt Hamer,
`Department of Neurosurgery, Academic Medical Center, Am-
`sterdam, The Netherlands), the xenograft cell line IGRG121 (a gift
`of Dr. B. Geoerger, Institut Gustave Roussy, Villejuif, France).
`Genomic DNA of the cell lines A1847, DU145, JAMA2, MCF7,
`ME180, MSTO-211H, NCI-H1299, NCI-H69, OE19, OE33,
`OVCA433, SCC9, SKCO1, and ZR-75-1 was provided by Dr. F.
`Di Nicolantonio (OncoGenomics Center, Institute for Cancer
`Research and Treatment, Italy). DNA from other cell lines was
`derived from our own laboratories.
`Genomic DNA was isolated as previously described (Balakrish-
`nan et al., 2007). PCR primers
`for
`the genomic region
`corresponding to IDH1 (NM_005896.2) exon 4, which encodes
`codon R132, and the flanking intronic sequences,
`including
`splicing donor and acceptor regions were designed using Primer 3
`(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The
`0
`0
`primers (forward 5
`-AATGAGCTCTATATGCCATCACTG-3
`, re-
`0
`0
`0
`verse 5
`-TTCATACCTTGCTTAATGGGTGT-3
`and sequence 5
`-
`0
`GCCATCACTGCAGTTGTAGGTTA-3
`) were synthesized by In-
`vitrogen/Life Technologies, Inc. (Paisley, England). PCRs were
`
`performed in 96-well formats in 10 ml reaction volumes, contain-
`ing 0.25 mmol/l deoxynucleotide triphosphates, 1 mmol/l each of
`the forward and reverse primers, 6% DMSO, 1  PCR buffer,
`1 ng/ml DNA, and 0.05 unit/ml Platinum Taq (Invitrogen/Life
`Technologies). A touchdown PCR program was used for PCR
`amplification (Peltier Thermocycler, PTC-200, MJ Research, Bio-
`Rad Laboratories, Inc., Italy).
`PCR conditions were as follows: 941C for 2 min; three cycles of
`941C for 15 sec, 641C for 30 sec, 701C for 30 sec; three cycles
`of 941C for 15 sec, 611C for 30 sec, 701C for 30 sec; three cycles of
`941C for 15 sec, 581C for 30 sec, 701C for 30 sec; and 35 cycles of
`941C for 15 sec, 571C for 30 sec, and 701C for 30 sec, followed by
`701C for 5 min, and 121C thereafter. PCR products were purified
`using AMPure (Agencourt Bioscience Corp., Beckman Coulter
`S.p.A, Milan, Italy). Cycle sequencing was carried out using BigDye
`Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster
`City, CA) with an initial denaturation at 971C for 3 min, followed
`by 28 cycles of 971C for 10 sec, 501C for 20 sec, and 601C for 2 min.
`Sequencing products were purified using CleanSeq (Agencourt
`Bioscience, Beckman Coulter) and analyzed on a 3730 DNA
`Analyzer, ABI capillary electrophoresis system (Applied Biosys-
`tems). Sequence traces were analyzed using the Mutation Surveyor
`software package (SoftGenetics, State College, PA).
`tumor
`A total of 756 PCR products, spanning 367 kb of
`genomic DNA, were generated and subjected to direct sequencing.
`Changes previously described as SNPs were excluded from further
`analyses (http://www.ensembl.org/index.html). To ensure that the
`observed mutations were not PCR or
`sequencing artifacts,
`amplicons were independently reamplified and resequenced in
`the corresponding tumors. All verified tumor changes were
`resequenced in parallel with the matched normal DNA to
`distinguish between somatic mutations and SNPs not previously
`described. For samples in which mutations were found, matching
`between germ-line and tumor DNA was verified by direct
`sequencing of 26 single nucleotide polymorphism (SNP) at 24
`loci (data not shown), to ensure that the observed changes are
`somatic mutations. Nucleotide and amino acid numbering uses
`the A of the ATG translation initiation start site (codon 1) as
`nucleotide 11, based on reference sequence NM_005896.2.
`The Fisher’s exact test (http://www.langsrud.com/fisher.htm)
`was used to determine the tissue specificity of IDH1R132
`mutations
`in HGG. In this test, the absence of mutations of different tumor
`types was compared with the number of mutations found in HGG
`samples.
`
`Results and Discussion
`We determined the occurrence of IDH1R132
`sequence variants in
`a panel of 672 tumor samples. These included 113 HGG samples
`(109 GBM, 2 anaplastic astrocytoma, and 2 anaplastic oligoden-
`droglioma), 25 GIST, 23 melanoma, 34 bladder cancer, 127 breast
`cancer, 128 colorectal cancer, 107 lung cancer, 46 ovarian cancer, 4
`prostate cancer, 42 thyroid cancer, and 23 PDAC specimens
`(Table 1). In addition, a panel consisting of 84 cell lines from
`multiple tumor lineages was screened for IDH1R132
`mutations.
`Out of the 756 samples analyzed, 23 displayed heterozygous
`the IDH1 gene. Strikingly,
`mutations at position R132 of
`mutations were only found in HGG (23 out of 113 samples
`corresponding to 20%; see Table 1). In agreement with previous
`results (Parsons et al., 2008), the most common change detected
`in our GBM tumor database is the IDH1p:R132H mutation. In
`addition to the reported p.R132 H and p.R132S variants, we
`detected three novel heterozygous somatic mutations affecting
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`Examples of novel somatic IDH1R132 mutations identified in GBM. Top, chromatogram of the sequence of a tumor sample; bottom,
`Figure 1.
`chromatogram of the matched normal. Arrows, location of missense somatic mutations. Nucleotide and amino acid alterations are below the
`traces. The nucleotide numbering uses the A of the ATG translation initiation start site as nucleotide 11, based on reference sequence
`NM_005896.2. Numbers above the sequences are part of the software output. A: p.R132G mutation; B: p.R132L mutation.
`
`residue R132 (p.R132C, p.R132G, and p.R132L) in GBM (Fig. 1,
`Table 2). As previously reported, most of the mutations were
`detected in glioblastomas. However, we also found a single
`mutated anaplastic oligodendroglioma. None of the other human
`solid tumor types displayed IDH1R132
`variants, and this was in
`most cases statistically significant (see Table 1). Although one
`colorectal cancer sample has been previously described to have an
`IDH1p:R132C allele (Sjo¨blom et al., 2006),
`in our set of 128
`colorectal cancer samples R132 variants were never detected.
`Cancer cell lines represent unique tools for multiple aspects of
`biomedical research including the evaluation of the functional
`relevance of cancer alleles. We therefore searched for mutations at
`position R132 of IDH1 in a panel of cancer cell lines, including 20
`astrocytoma (Table 3 and Supp. Table S1). None of the cell lines
`displayed IDH1R132
`variants. However, we found two previously
`unreported IDH1 alleles in three cell lines: p.V71I was detected in
`the plasma cell myeloma line RPMI-8226, while p.G97D was
`found in the colorectal cancer cell lines DLD-1 and HCT-15. These
`two colorectal cancer cell lines are suggested to be genetically
`identical, and therefore may be derived from the same patient
`(Chen et al., 1995). As matched normal samples are not available
`for these tumor cell lines, we cannot assess whether the nature of
`these mutations is somatic. Neither the p.V71I nor the p.G97D
`variants have been reported previously as SNPs (http://www.
`ensembl.org/index.html). Considering that we did not find these
`alleles in any of the 672 tumor samples and 84 cell lines that we
`sequenced, we suspect that they are either very rare SNPs or novel
`IDH1 somatic changes. Compared to the frequency (20%) that we
`found in HGG samples, the lack of IDH1 mutations in our panel
`of 20 high-grade astrocytoma cell
`lines appears statistically
`significant (p-value 5 0.024, Fisher’s exact test). It is possible,
`however, that GBM cell lines are predominantly derived from
`primary GBM tumors, thus explaining our results.
`GBM, WHO grade IV with predominant astrocytic differentia-
`tion, is the most common and most aggressive primary brain
`tumor (Louis et al., 2007). Most glioblastoma manifest rapidly de
`novo, without recognizable precursor lesions. These so-called
`primary GBMs typically present in middle age to elderly patients
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`
`with a brief clinical history and show rapid progression and short
`survival time (Ohgaki and Kleihues, 2007; Scherer, 1940). In
`contrast, secondary GBMs are typically seen in younger patients,
`with a history of epilepsy caused by low-grade gliomas, which in
`years progress to GBM (Ohgaki and Kleihues, 2007; Scherer,
`1940). Secondary GBMs are rare (5%) in comparison to primary
`GBM (Ohgaki et al., 2004), and can only be diagnosed with
`clinical (neuroimaging) or histological evidence of evolution from
`a less malignant astrocytoma (Ohgaki and Kleihues, 2007). Both
`subtypes are considered histopathologically indistinguishable.
`However, the classification in primary and secondary is nicely
`reflected by molecular mechanisms. Primary GBMs have a high
`rate of EGFR alterations, MDM2 duplications, PTEN mutations,
`and homozygous P16INK4A
`deletions, whereas TP53 mutations are
`most prevalent in secondary GBMs (Ohgaki and Kleihues, 2007;
`IDH1 mutations
`Ohgaki
`et al., 2004). We observed the
`predominantly in secondary GBM (11 of 94 vs. 11 of 15, p-
`value 5 0.0000016, Fisher’s exact test; see Table 1), in accordance
`with the results of Parsons and colleagues (2008). In addition to
`the IDH1p:R132C=G=H=L mutations in GBM, we identified an
`IDH1p:R132S mutation in an anaplastic oligodendroglioma sample.
`Interestingly, GBM patients with an IDH1R132
`mutation have been
`reported to have a better survival (Parsons et al., 2008). No
`information on IDH1 mutations in low-grade gliomas is available
`thus far; therefore, assessment of whether lower grade gliomas
`display IDH1R132
`mutations and if they have a survival advantage
`are critical questions that should be addressed.
`In conclusion, our data support the evidence that IDH1 is a
`pivotal GBM cancer gene mutated predominantly in secondary
`glioblastomas. The identification of three novel mutations in
`IDH1 affecting amino acid R132 may allow further structural and
`functional analysis of the function of this residue on the catalytic
`activity of isocitrate dehydrogenase 1. Our most relevant finding
`entails the unique and striking tissue-specific pattern of the
`IDH1R132 mutations in human solid cancer. The tissue specificity
`of cancer mutations has been observed in multiple cancer genes
`(e.g., APC, AKT1) (Bleeker et al., 2008a; Bleeker et al., 2008b).
`Why some genes are mutated in specific tumor types remains an
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`unsettled issue whose solution will be relevant for basic and
`clinical cancer research. As IDH1 is involved in a specific
`metabolic pathway, its mutations may potentially be exploited
`for therapeutic purposes. However, to therapeutically challenge
`the IDH1 cancer variants it must first be assessed whether they
`functionally operate as oncogenes or tumor suppressor genes. The
`fact that we and others only found heterozygous mutations at one
`specific IDH1 residue involved in its catalytic activity, strongly
`suggests that these mutations could activate IDH1 in a pro-
`oncogenic (dominant) fashion in cancer cells. Studies revealing
`the functional role of the IDH1R132
`mutations are vital to confirm
`this hypothesis and to provide insights in the potential of mutated
`IDH1 as therapeutic target.
`
`Acknowledgments
`
`The authors thank Dr. C. Zanon for help with sequencing, and Dr. S.
`Thorlacius and Dr. F. Di Nicolantonio for critical reading of the manuscript.
`F.B. is supported by a Netherlands Genomic Initiative Fellowship.
`
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