Mutation Analysis of IDH1 in Paired Gliomas Revealed IDH1 Mutation Was Not Associated with Malignant Progression but Predicted Longer Survival

et al. (2013) Mutation Analysis of IDH1 in Paired Gliomas Revealed IDH1 Mutation Was Not Associated with Malignant Progression but Predicted Longer Survival. PLoS ONE 8(6): e67421. doi:10.1371/journal.pone.0067421 Mutation Analysis of IDH1 in Paired Gliomas Revealed IDH1 Mutation Was Not Associated with Malignant Progression but Predicted Longer Survival Yu Yao 0 Aden Ka-Yin Chan 0 Zhi Yong Qin 0 Ling Chao Chen 0 Xin Zhang 0 Jesse Chung-Sean Pang 0 Hiu Ming Li 0 Yin Wang 0 Ying Mao 0 Ho-Keung NG 0 Liang Fu Zhou 0 Svetlana Kotliarova, NIH/NCI, United States of America 0 1 Department of Neurosurgery, Huashan Hospital, Fudan University , Shanghai , China , 2 Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, 3 Shenzhen Research Institute, The Chinese University of Hong Kong , Shenzhen , China , 4 Department of Neuropathology, Huashan Hospital, Fudan University , Shanghai , China Recurrence and progression to higher grade lesions are characteristic behaviorsof gliomas. Though IDH1 mutation frequently occurs and is considered as an early event in gliomagenesis, little is known about its role in the recurrence and progression of gliomas. We therefore analysed IDH1 and IDH2 statusat codon 132 of IDH1 and codon 172 of IDH2 by direct sequencing and anti-IDH1-R132H immunohistochemistry in 53 paired samples and their recurrences, including 29 lowgrade gliomas, 16 anaplastic gliomas and 8 Glioblastomas. IDH1/IDH2 mutation was detected in 32 primarytumors, with 25 low- grade gliomas and 6 anaplastic gliomas harboring IDH1 mutation and 1 low- grade glioma harboring IDH2 mutation. All of the paired tumors showed consistent IDH1 and IDH2 status. Patients were analyzed according to IDH1 status and tumor-related factors. Malignant progression at recurrence was noted in 22 gliomas and was not associated with IDH1 mutation. Survival analysis revealed patients with IDH1 mutated gliomas had a significantly longer progression-free survival (PFS) and overall survival (OS). In conclusion, this study demonstrated a strong tendency of IDH1/IDH2 status being consistent during progression of glioma. IDH1 mutation was not a predictive marker for malignant progression and it was a potential prognostic marker for gliomas of Chinese patients. - Funding: This work was supported by The Project for Science and Technology Commission of Shanghai Municipality Grant (No. 10JC1402200 to L.F.Z.), National Natural Science Foundation of China (81172412 to H.K.N.), Shanghai Talents Development Funds (No. 2011063 to Y.Y.), The Grant from Ministry of Health of China (W2012FZ003 to Z.Y.Q.), Wu Jieping medical foundation for clinical research of special fund project (No. 320.6700.1162 to Y.Y.), The Project for National 985 Engineering of China (No.985III-YFX0102 to Y.M.). Science and Technology Commission of Shanghai Municipality Grant (No. 10JC1402200 to L.F.Z.) had a role in study design; National Natural Science Foundation of China (81172412 to H.K.N.) had a role in experiment, data collection and analysis; Shanghai Talents Development Funds (No. 2011063 to Y.Y.) have a role in decisions to follow up, publish and prepare the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Gliomas are the most common primary brain tumors, accounting for 80% of malignant central nervous system neoplasms [1].Recent genome-wide mutational analysis has demonstrated that the incidence of IDH1 mutations in gliomas ranges from 5% in primary glioblastoma (GBM) to 70% in anaplastic astrocytomas (AA) and 80% in secondary GBM [26]. Patients with high-grade astrocytomas with IDH1 mutations were reported to have a better survival [6]. The IDH1 gene is located on 2q33.3 and its mutation has been described in a very restricted number of human cancers including gliomas [3,7,8].The most common IDH1 mutation is a heterozygous missense mutation with a change of guanine to adenine at position 395 (G395A), leading to the replacement of arginine by histidine at codon132 (IDH1-R132H) at the enzymatic active site [10]. IDH1 mutation has been shown to occur in early stage of gliomagenesis [5]. The pathogenesis of IDH1-R132Hrelated tumorigenesis is rapidly being elucidated. Not only does loss of function occur with reduced production of a-ketoglutarate (a-KG) from isocitrate, the R132H mutation also confers a gain of function to the mutant IDH1, which converts a-KG to 2hydroxyglutarate (2-HG) [11]. Accumulation of this oncometabolite induce sextensive DNA hypermethylation, leading to genomewide epigenetic changes and predisposing cells toward neoplastic transformation [12]. In spite of all the studies, the role of IDH1 mutation in the recurrence of gliomas is unknown. There have been few studies in which paired gliomas at primary presentation and recurrence were studied by molecular means. In the present study, we investigated the mutation status of IDH1 and IDH2 in 53pairs of primary and recurrent gliomas. All pairs showed consistent IDH1/IDH2status.Correlation analysis with clinicopathological parameters revealed thatIDH1 mutation was not associated with malignant progression but was a potential prognostic marker for progressionfree survival (PFS) and overall survival (OS) in astrocytomas. G G BM tao Patients and Methods Ethics Statement This study was approved by the Ethics Committee of Shanghai Huashan Hospital and the New Territories East Cluster-Chinese University of Hong Kong Ethics Committee. Patients and Tissue Samples Records of patients with glioma diagnosed in the Department of Neurosurgery, Huashan hospital (Shanghai, China) and Department of Anatomical and Cellular Pathology, Prince of Wales Hospital (Hong Kong) between 1990 and 2011 were reviewed. 53paired cases were retrieved where formalin-fixed paraffin embedded (FFPE) tissues were available from primary presentations and recurrences (Table S1). Haematoxylin &eosin (H&E) stained sections of each tumor were reviewed and graded according to the 2007 WHO classification of tumors of central nervous system. Mutation Analysis of IDH1/IDH2 Mutational hotspots of IDH1at codon 132 and IDH2 at codon 172 were evaluated by direct sequencing. Representative tumor area scrapped off from dewaxed sections into microfuge tubes were resuspended in 10 mMTris-HCl buffer, pH 8.5. Proteinase K was added to a final concentration of 2g/l and the mixture was incubated at 55uC for 2 hours and then at 98uC for 10 min. The PCR mixture of 10 ml volume contained 12 ml of crude cell lysate, 10 mMTris-HCl (pH 8.3), 50 mMKCl, 2.5 mM MgCl2, 0.2 mM of each deoxyribonucleoside triphosphate, 0.4 mM of each primer (IDH1-F: 59-CGGTCTTCAGAGAAGCCATT-39 and IDH1-R: 59-CACATTATTGCCAACATGAC-39; IDH2-F: 59-AGCCCATCATCTGCAAAAAC-39 and IDH2-R: 59CTAGGCGAGGAGCTCCAGT-39) [5,9] and 0.2 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Hong Kong). PCR was initiated at 95u (...truncated)