Int. J. Cancer: 125, 353–355 (2009)
`' 2009 UICC
`
`Mutational analysis of IDH1 codon 132 in glioblastomas
`and other common cancers
`Mi Ran Kang1, Min Sung Kim1, Ji Eun Oh1, Yoo Ri Kim1, Sang Yong Song2, Seong Il Seo3,
`Ji Youl Lee4, Nam Jin Yoo1 and Sug Hyung Lee1*
`1Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea
`2Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
`3Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
`4Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Korea
`
`Missense somatic mutations in IDH1 gene affecting codon 132
`have recently been reported in glioblastoma multiforme (GBM)
`and other gliomas. The recurrent nature of the IDH1 mutations in
`the same amino acid strongly suggests that the mutations may
`play important roles in the pathogenesis of glial tumors. The aim
`of this study was to see whether the IDH1 codon 132 mutations
`occur in other human cancers besides glial tumors. We also
`attempted to confirm the occurrence of the IDH1 mutations in
`GBM of Korean patients. We have analyzed 1,186 cancer tissues
`from various origins, including carcinomas from breast, colon,
`lung, stomach, esophagus, liver, prostate, urinary bladder, ovary,
`uterine cervix, skin and kidney, and malignant mesotheliomas,
`primary GBM, malignant meningiomas, multiple myelomas and
`acute leukemias by single-strand conformation polymorphism
`analysis. We found four IDH1 codon 132 mutations in the GBM
`(4/25; 16.0%), two in the prostate carcinomas (2/75; 2.7%) and
`one in the B-acute lymphoblastic leukemias (B-ALL) (1/60; 1.7%),
`but none in other cancers. The IDH1 mutations consisted of five
`p.R132H and two p.R132C mutations. The data indicate that
`IDH1 codon 132 mutations occur not only in GBM, but also in
`prostate cancers and B-ALL. This study suggests that despite the
`infrequent incidence of the IDH1 mutations in prostate cancers
`and B-ALL, mutated IDH1 could be therapeutically targeted in
`these cancers and in glial tumors with the IDH1 mutations.
`' 2009 UICC
`
`Key words: IDH1; glioblastoma multiforme; mutation; IDH1 codon
`132; cancer
`
`For the comprehensive elucidation of genetic alterations in glio-
`blastoma multiforme (GBM), Parsons et al.1 recently analyzed
`20,661 genes (approximately two-thirds of total human genes) by
`a direct DNA sequencing method. In GBM tissues, they identified
`685 genes that contained at least one nonsilent somatic muta-
`tion(s). In addition to the genes with known mutations in GBM
`such as TP53, EGFR, PTEN, NF1, RB1 and PIK3CA, two genes
`isocitrate dehydrogenase 1 (IDH1) and phosphoinositide-3-kinase
`regulatory subunit 1 (PIK3R1) harbored mutations at high inci-
`dences.1 Somatic point mutations of IDH1 were detected in 12 of
`105 GBM (11.4%). Of note, all of the 12 mutations were predicted
`to substitute an Arg residue in position 132 of amino acid sequen-
`ces. A following study using various brain tumors detected the
`IDH1 codon 132 mutations not only in GBM (primary and second-
`ary), but also in other glial tumors.2 Despite the high incidence of
`the IDH1 mutations in the brain tumors, the functional roles of the
`mutations in cancer development remain unknown.
`One of the main concerns in cancer genetics is as to whether
`any mutation found in cancer is specific to few cancer types or is
`widespread in many cancer types. For example, EGFR and JAK2
`mutation is specific to few cancer types,3,4 whereas K-RAS and
`TP53 mutations are common to many cancer types.5,6 To see if
`any other types of human cancers besides glial tumors carry the
`IDH1 codon 132 mutations, we have analyzed the IDH1 gene in
`various types of human cancers in this study.
`
`Material and methods
`
`Cancer tissues (1,186) from Korean patients (carcinomas from
`breast, colon, lung, stomach, esophagus, liver, prostate, urinary
`
`Publication of the International Union Against Cancer
`
`bladder, ovary, uterine cervix, skin and kidney, and malignant
`mesotheliomas, primary GBM, malignant meningiomas, multiple
`myelomas and acute leukemias) were used for this study (Table I).
`All of the cancers analyzed were primary cancers, but not meta-
`static cancers. We did not include cell lines in this study. For the
`solid cancers, malignant cells and normal cells were selectively
`procured from hematoxylin and eosin-stained slides using a 301/2
`gauge hypodermic needle affixed to a micromanipulator, as
`described previously.7 Approval for this study was obtained from
`the Catholic University of Korea, College of Medicine’s institu-
`tional review board.
`Up to now, all of the IDH1 mutations have been detected at nu-
`cleotide sequences 394 or 395 in exon 4, which would result in
`amino acid substitutions at 132.1,2 Thus, we analyzed a part of the
`exon 4 of IDH1 gene by polymerase chain reaction (PCR)-based
`single-strand conformation polymorphism (SSCP)
`analysis.
`Genomic DNA each from tumor cells and normal cells of
`the same patients were amplified by PCR with one primer pair
`(50-AAACAAATGTGGAAATCACC-30
`and 50-TGCCAACAT
`GACTTACTTGA-30; product size 166 base pairs). Radioisotope
`([32P]dCTP) was incorporated into the PCR products for detection
`by autoradiogram. Other procedures of the PCR-SSCP were
`described in our previous studies.7–9 After SSCP, direct DNA
`sequencing reactions were performed in the cancers with the mo-
`bility shifts in the SSCP according to the manufacturer’s recom-
`mendation (ABI Prism Genetic Analyzer, Applied Biosystem,
`Foster City, CA). As potential positive controls for the SSCP, we
`included GBM tissues with known IDH1 mutations.
`
`Results and discussion
`
`PCR-SSCP analysis of the exon 4 of IDH1 gene in the 1,186
`cancers identified aberrant bands in seven cancers (Fig. 1). There
`was no visible aberrant band in the other 1,179 cancers. Direct
`DNA sequencing analysis of the PCR products in the seven can-
`cers [four GBMs, two prostate cancers and one B-acute lympho-
`blastic leukemia (B-ALL)] with the aberrant SSCP bands led to
`the identification of seven IDH1 mutations (Fig. 1; Table II). None
`of the normal samples from the same patients showed evidence of
`mutations by the SSCP and direct DNA sequencing (Fig. 1), indi-
`cating the mutations had risen somatically. The IDH1 mutations
`consisted of five c.395G>A (p.R132H) and two c.394C>T
`(p.R132C; Table II). We repeated the experiments twice, includ-
`ing PCR, SSCP and sequencing analysis to ensure the specificity
`of the results and found that the data were consistent (data not
`shown).
`
`Grant sponsor: Korea Science and Engineering Foundation (KOSEF);
`Grant number: R01-2008-000-10014-0.
`*Correspondence to: Department of Pathology, College of Medicine,
`The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-
`701, Korea. Fax: 182-2-537-6586. E-mail: suhulee@catholic.ac.kr
`Received 15 December 2008; Accepted after revision 10 February 2009
`DOI 10.1002/ijc.24379
`Published online 23 February 2009 in Wiley InterScience (www.interscience.
`wiley.com).
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`354
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`KANG ET AL.
`
`the seven cancers with the IDH1
`The SSCP of all of
`mutations at
`the mutation sites showed both wild-type and
`aberrant bands (Fig. 1a), and direct sequencing analysis also
`
`TABLE I – IDH1 MUTATIONS IN 1,185 CANCERS
`
`Type of cancers
`
`Number
`of cancers
`
`Primary GBM
`Prostate carcinoma
`B-ALL
`T-ALL
`Acute myelogenous
`leukemia
`Multiple myeloma
`Malignant meningioma
`Malignant mesothelioma
`Non-small cell lung cancer
`Gastric carcinoma
`Colorectal carcinoma
`Breast carcinoma
`Hepatocellular carcinoma
`Hepatoblastoma
`Esophageal squamous
`cell carcinoma
`Urothelial carcinoma
`Uterine cervical carcinoma
`Ovarian carcinoma
`Squamous cell carcinoma,
`skin
`Renal cell cracinoma
`
`25
`75
`60
`26
`100
`
`30
`29
`6
`179
`101
`97
`94
`81
`56
`71
`
`28
`10
`85
`19
`
`14
`
`Wild
`type
`
`21
`73
`59
`26
`100
`
`30
`29
`6
`179
`101
`97
`94
`81
`56
`71
`
`28
`10
`85
`19
`
`14
`
`IDH1 codon 132
`
`Mutation Mutation
`(%)
`
`4
`2
`1
`0
`0
`
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`
`0
`
`16
`2.7
`1.7
`0
`0
`
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`
`0
`
`revealed both mutant and wild-type sequences (Fig. 1b). We
`used microdissected tissues that had purity of cancer cells
`over 90%.7 Thus, both SSCP and direct sequencing data indi-
`the IDH1 mutations may be heterozygous in the
`cate that
`seven cancers.
`We compared the incidences of the IDH1 mutations in primary
`GBM between the previous (7/99)1 and our data (4/25). There was
`no statistical difference between them (Fisher’s exact test, p >
`0.05). The prostate carcinomas used in the present study consisted
`of 55 TNM II, 19 TNM III and 1 TNM IV cancers. According to
`the Gleason score, the prostate carcinomas were graded as Glea-
`son score 6 (n 5 18), Gleason score 7 (n 5 47), Gleason score 8
`(n 5 8) and Gleason score 9 (n 5 2). However, there was not any
`significant association of the IDH1 mutations either with the Glea-
`son scores or the TNM stages (v2 test, p > 0.05).
`Recurrent genetic alterations have been used as therapeutic tar-
`gets in cancer treatment. For example, recurrent mutations of
`EGFR in lung cancers, BCR/ABL translocations in chronic my-
`elogenous leukemias and HER2 amplifications in breast cancers
`are being used as therapeutic targets of Gefitinib (Iressa), Imanitib
`(Gleevec) and Trastzumab (Herceptin), respectively.4,10–12 Recur-
`rent IDH1 mutations in a specific amino acid (Arg 132) suggested
`a possibility that targeting of the IDH1 mutation could be used in
`the treatment of cancers harboring the mutations as in the cases of
`EGFR, BCR/ABL and HER2.4,10–12 In the current study, we found
`that prostate cancers and B-ALL harbored the IDH1 codon 132
`mutations. Also, we confirmed the occurrence of the IDH1 muta-
`tion in the GBM of Korean patients. Despite the low frequency of
`the IDH1 mutations in these cancers compared with that of glial
`
`FIGURE 1 – Mutations of IDH1 gene in prostate cancers and B-ALL. SSCP (a) and DNA sequencing analysis (b) of DNA from tumor (Lane
`T) and normal tissues (Lane N). (a) Arrows (Lane T) indicate aberrant bands compared with the SSCP from normal tissues (N). SSCPs of DNA
`from tumors (T) show wild-type bands and additional aberrant bands. (b) Direct DNA sequencing analyses from prostate cancers #5 (left) and
`#27 (middle) and B-ALL #29 (right). There are nucleotide substitutions (red box) in tumor tissues (T) as compared with normal tissues (N).
`[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
`
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`IDH1 MUTATION IN CANCERS
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`355
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`TABLE II – SUMMARY OF THE IDH1 MUTATIONS IN THE CANCERS
`
`Case no.
`
`Type of cancers
`
`GBM 6
`GBM 23
`GBM 27
`GBM 31
`PS 5
`PS 27
`Leu 29
`
`Primary GBM
`Primary GBM
`Primary GBM
`Primary GBM
`Prostate adenocarcinoma
`Prostate adenocarcinoma
`B-ALL
`
`Nucleotide change
`(Predicted amino acid change)
`
`c.395G>A (p.R132H)
`c.395G>A (p.R132H)
`c.395G>A (p.R132H)
`c.395G>A (p.R132H)
`c.395G>A (p.R132H)
`c.394C>T (p.R132C)
`c.394C>T (p.R132C)
`
`Other clinicopathologic
`characteristics
`
`–
`–
`–
`–
`TNM stage II, Gleason score 7
`TNM stage III, Gleason score 8
`BRAF mutation (c.486G>A), no cytogenetic abnormality
`
`tumors, the IDH1 mutation may play an important role in the de-
`velopment of the cancers with the mutations, given that the IDH1
`mutations provide cancer-related functions with the affected cells.
`Also, the data suggest a possibility that not only glial tumors, but
`also prostate and B-ALL should be considered as candidate cancer
`types for future therapies targeting the IDH1 mutations. Recently,
`Bleeker et al.13 analyzed the IDH1 mutation in a large series of
`tumors, and observed comparable results to our data. They found
`no IDH1 mutation in other tumors besides GBM.
`To date, there have been six types of the IDH1 codon 132 muta-
`tions (p.R132H, p.R132C, p.R132S, p.R132G, p.R132L and
`p.R132V) detected in human cancers.1,2 In glial tumors, p.R132H
`is the most common IDH1 mutation and p.R132C is the second
`one.1,2 In agreement with these, the IDH1 mutations detected in
`our study were either p.R132H or p.R132C. However, functional
`role of the IDH1 mutations in cancer pathogenesis depending on
`the subtypes remains to be clarified.
`IDH1 is an enzyme that catalyzes the oxidative decarboxylation
`of isocitrate into a-ketoglutarate utilizing either NAD or NADP as
`cosubstrates.14,15 IDH1 is mainly involved in metabolic processes
`and its roles in cancer biology are largely unknown. Thus, it is
`
`crucial to discover the functions of mutant IDH1 in cancer devel-
`opment. However, before the identification of functions, its nor-
`mal physiological functions must be found out first. One of the
`cancer-related functions of IDH1 is the cellular control of oxida-
`tive damages.16 Whether alteration of
`its oxidative damage
`response by the IDH1 mutations is crucial in cancer development,
`and/or whether alterations of other unidentified functions are cru-
`cial, should be further clarified. In addition, analysis of alterations
`of tissue expression of IDH1 gene together with the IDH1 muta-
`tions could further elucidate roles of IDH1 in cancers.
`In summary, this study identified that few types of human
`cancers harbored IDH1 codon 132 mutations, suggesting some
`overlap of pathogenesis among glial tumors, prostate cancers and
`B-ALL. Despite the infrequent incidence of the IDH1 mutations
`in prostate cancers and B-ALL, mutated IDH1 could also be thera-
`peutically targeted in these cancers with the IDH1 mutations.
`
`Acknowledgement
`
`The prostate cancer tissues were supplied from ‘The Prostate
`Bank of KoreaÕ supported by KOSEF.
`
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