Mutational analysis of IDH1 codon 132 in glioblastomas and other common cancers Mi Ran Kang 1 , Min Sung Kim 1 , Ji Eun Oh 1 , Yoo Ri Kim 1 , Sang Yong Song 2 , Seong Il Seo 3 , Ji Youl Lee 4 , Nam Jin Yoo 1 and Sug Hyung Lee 1 * 1 Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 3 Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 4 Department 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 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 (5 0 -AAACAAATGTGGAAATCACC-3 0 and 5 0 -TGCCAACAT GACTTACTTGA-3 0 ; product size 166 base pairs). Radioisotope ([ 32 P]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). Int. J. Cancer: 125, 353–355 (2009) ' 2009 UICC Publication of the International Union Against Cancer