Comparative study of IDH1 mutations in gliomas by high resolution melting analysis, immunohistochemistry and direct DNA sequencing

Li,Juan; Zhang,Haiyan; Wang,Li; Yang,Chuanhong; Lai,Huangwen; Zhang,Wei; Chen,Xiaodong; Wang,Jie

doi:10.3892/mmr.2015.3987

Comparative study of IDH1 mutations in gliomas by high resolution melting analysis, immunohistochemistry and direct DNA sequencing

Authors:
- Juan Li
- Haiyan Zhang
- Li Wang
- Chuanhong Yang
- Huangwen Lai
- Wei Zhang
- Xiaodong Chen
- Jie Wang
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Affiliations: Department of Biochemistry and Molecular Biology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong 510006, P.R. China, Department of Medical Research, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong 510010, P.R. China, Department of Pathology, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong 510010, P.R. China
Published online on: June 23, 2015 https://doi.org/10.3892/mmr.2015.3987
Pages: 4376-4381

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Abstract

Patients with glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than those with gliomas with wild‑type IDH1. IDH1 analysis has become part of the standard diagnostic procedure and a promising tool used for stratification in clinical trials. The present study aimed to compare high resolution melting (HRM) analysis, immunohistochemistry (IHC) and direct DNA sequencing for the detection of IDH mutations in gliomas. Fifty‑one formalin‑fixed paraffin‑embedded tumor samples were selected. For the HRM analysis and direct DNA sequencing, DNA was extracted from the tissues. For IHC, sections were stained with an anti‑IDH1‑R132H specific antibody. The HRM analysis method identified 33 cases of IDH1 gene mutations, and all mutations occurred at the R132H site. There were 33 cases of IDH1 gene mutations found by IHC, which was consistent with that identified using the HRM analysis method. However, only 30 IDH1 samples were confirmed by sequencing, in which mutations occurred at the IDH1 exon 4 R132H site. No mutation was detected in the other three of these 33 cases (two grade II oligodendroglioma and one grade II diffuse astrocytoma) by sequencing, while IHC was positive for IDH1‑R132H. The results showed that the mutation detection rate was not identified to be significantly different (P=0.250) when determined by the HRM analysis method or by direct DNA sequencing, as the concordant rate between the two methods was high (κ=0.866). The HRM analysis method in glioma IDH1 gene mutation detection has advantages of high sensitivity, good repeatability, simple operation and accurate results. It provides a novel method for detecting mutations of the IDH1 gene in paraffin embedded tissue samples of clinical glioma. Related to a small amount of sample, there was no evidence showing that HRM analysis method is superior to IHC. Direct DNA sequencing, HRM analysis and IHC results were consistent; however, HRM and IHC are more sensitive than direct DNA sequencing in identifying the IDH1‑R132H mutation.

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1	Hartmann C, Meyer J, Balss J, Capper D, Mueller W, Christians A, Felsberg J, Wolter M, Mawrin C, Wick W, et al: Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: A study of 1010 diffuse gliomas. Acta Neuropathol. 118:469–474. 2009. View Article : Google Scholar : PubMed/NCBI
2	Gravendeel LA, Kloosterhoof NK, Bralten LB, van Marion R, Dubbink HJ, Dinjens W, Bleeker FE, Hoogenraad CC, Michiels E, Kros JM, et al: Segregation of non-p.R132H mutations in IDH1 in distinct molecular subtypes of glioma. Hum Mutat. 31:E1186–E1199. 2010. View Article : Google Scholar : PubMed/NCBI
3	Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, et al: IDH1 and IDH2 mutations in gliomas. N Engl J Med. 360:765–773. 2009. View Article : Google Scholar : PubMed/NCBI
4	Watanabe T, Nobusawa S, Kleihues P and Ohgaki H: IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am J Pathol. 174:1149–1153. 2009. View Article : Google Scholar : PubMed/NCBI
5	Bralten LB, Kloosterhof NK, Balvers R, Sacchetti A, Lapre L, Lamfers M, Leenstra S, de Jonge H, Kros JM, Jansen EE, et al: IDH1 R132H decreases proliferation of glioma cell lines in vitro and in vivo. Ann Neurol. 69:455–463. 2011. View Article : Google Scholar : PubMed/NCBI
6	Lu C, Ward PS, Kapoor GS, Rohle D, Turcan S, Abdel-Wahab O, Edwards CR, Khanin R, Figueroa ME, Melnick A, et al: IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature. 483:474–478. 2012. View Article : Google Scholar : PubMed/NCBI
7	Nobusawa S, Watanabe T, Kleihues P and Ohgaki H: IDH1 mutations as molecular signature and predictive value of secondary glioblastomas. Clin Cancer Res. 15:6002–6007. 2009. View Article : Google Scholar : PubMed/NCBI
8	Sanson M, Marie Y, Paris S, Idbaih A, Laffaire J, Ducray F, El Hallani S, Boisselier B, Mokhtari K, Hoang-Xuan K, et al: Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol. 27:4150–4154. 2009. View Article : Google Scholar : PubMed/NCBI
9	Hartmann C, Hentschel B, Wick W, Capper D, Felsberg J, Simon M, Westphal M, Schackert G, Meyermann R, Pietsch T, et al: Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas and IDH1 mutation status accounts for the unfavourable prognostic effect of higher age: Implications for classification of gliomas. Acta Neuropathol. 120:707–718. 2010. View Article : Google Scholar : PubMed/NCBI
10	Van den Bent MJ, Hartmann C, Preusser M, Ströbel T, Dubbink HJ, Kros JM, von Deimling A, Boisselier B, Sanson M, Halling KC, et al: Interlaboratory comparison of IDH mutation detection. J Neurooncol. 112:173–178. 2013. View Article : Google Scholar : PubMed/NCBI
11	Capper D, Zentgraf H, Balss J, Hartmann C and von Deimling A: Monoclonal antibody specific for IDH1 R132H mutation. Acta Neuropathol. 118:599–601. 2009. View Article : Google Scholar : PubMed/NCBI
12	Kato Y, Jin G, Kuan CT, McLendon RE, Yan H and Bigner DD: A monoclonal antibody IMab-1 specifically recognizes IDH1 R132H, the most common glioma-derived mutation. Biochem Biophys Res Commun. 390:547–551. 2009. View Article : Google Scholar : PubMed/NCBI
13	Kaneko MK, Tian W, Takano S, Suzuki H, Sawa Y, Hozumi Y, Goto K, Yamazaki K, Kitanaka C and Kato Y: Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation. Biochem Biophys Res Commun. 406:608–613. 2011. View Article : Google Scholar : PubMed/NCBI
14	Kaneko MK, Morita S, Tsujimoto Y, Yanagiya R, Nasu K, Sasaki H, Hozumi Y, Goto K, Natsume A, Watanabe M, et al: Establishment of novel monoclonal antibodies KMab-1 and MMab-1 specific for IDH2 mutations. Biochem Biophys Res Commun. 432:40–45. 2013. View Article : Google Scholar : PubMed/NCBI
15	Kato Y, Natsume A and Kaneko MK: A novel monoclonal antibody GMab-m1 specifically recognizes IDH1-R132G mutation. Biochem Biophys Res Commun. 432:564–567. 2013. View Article : Google Scholar : PubMed/NCBI
16	Cui G, Ding H, Xu Y, Li B and Wang DW: Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population. Gene. 108–112. 2013. View Article : Google Scholar
17	Harle A, Lion M, Lozano N, Husson M, Harter V, Genin P and Merlin JL: Analysis of PIK3CA exon 9 and 20 mutations in breast cancers using PCR-HRM and PCR-ARMS: Correlation with clinicopathological criteria. Oncol Rep. 29:1043–1052. 2013.PubMed/NCBI
18	Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Siu IM, Gallia GL, et al: An integrated genomic analysis of human glioblastoma multiforme. Science. 321:1807–1812. 2008. View Article : Google Scholar : PubMed/NCBI
19	Johnson BE, Mazor T, Hong C, Barnes M, Aihara K, McLean CY, Fouse SD, Yamamoto S, Ueda H, Tatsuno K, et al: Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science. 343:189–193. 2014. View Article : Google Scholar :
20	Capper D, Weissert S, Balss J, Habel A, Meyer J, Jäger D, Ackermann U, Tessmer C, Korshunov A, Zentgraf H, et al: Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors. Brain Pathol. 20:245–254. 2010. View Article : Google Scholar
21	Agarwal S, Sharma MC and Jha P, Pathak P, Suri V, Sarkar C, Chosdol K, Suri A, Kale SS, Mahapatra AK and Jha P: Comparative study of IDH1 mutations in gliomas by immunohistochemistry and DNA sequencing. Neuro Oncol. 15:718–726. 2013. View Article : Google Scholar : PubMed/NCBI
22	Agarwal S, Sharma MC, Jha P, Pathak P, Suri V, Sarkar C, Chosdol K, Suri A, Kale SS, Mahapatra AK, et al: Comparative study of IDH1 mutations in gliomas by immunohistochemistry and DNA sequencing. Neuro Oncol. 15:718–726. 2013. View Article : Google Scholar : PubMed/NCBI
23	Kurian KM, Haynes HR, Crosby C, Hopkins K and Williams M: IDH mutation analysis in gliomas as a diagnostic and prognostic biomarker. Br J Neurosurg. 27:442–445. 2013. View Article : Google Scholar : PubMed/NCBI
24	Takano S, Tian W, Matsuda M, Yamamoto T, Ishikawa E, Kaneko MK, Yamazaki K, Kato Y and Matsumura A: Detection of IDH1 mutation in human gliomas: Comparison of immunohistochemistry and sequencing. Brain Tumor Pathol. 28:115–123. 2011. View Article : Google Scholar : PubMed/NCBI
25	Wittwer CT: High-resolution DNA melting analysis: Advancements and limitations. Hum Mutat. 30:857–859. 2009. View Article : Google Scholar : PubMed/NCBI
26	Lin J, Qian J, Yao DM, Li Y, Yang J, Chen Q, Chai HY, Xiao GF and Xu WR: Rapid and reliable detection of IDH1 R132 mutations in acute myeloid leukemia using high-resolution melting curve analysis. Clin Biochem. 44:779–783. 2011. View Article : Google Scholar : PubMed/NCBI
27	Takano S, Tian W, Matsuda M, Yamamoto T, Ishikawa E, Kaneko MK, Yamazaki K, Kato Y and Masumura A: Detection of IDH1 mutation in human gliomas: Comparison of immunohisto-chemistry and sequencing. Brain Tumor Pathol. 28:115–123. 2011. View Article : Google Scholar : PubMed/NCBI