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ORIGINAL ARTICLE
Year : 2012  |  Volume : 8  |  Issue : 4  |  Page : 598-601

Immunohistochemical expression of IDH1 in gliomas: A tissue microarray-based approach


1 National Institute of Pathology, ICMR, New Delhi, India
2 Department of Neurosurgery, Safdajung Hospital, New Delhi, India

Date of Web Publication29-Jan-2013

Correspondence Address:
Avninder Singh
213-Pocket B Sukhdev Vihar, New Delhi - 110025
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.106567

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 > Abstract 

Background: Mutations in the gene encoding isocitrate dehydrogenase (IDH1) have been reported in gliomas. This study analyses a series of 184 glioma cases in a tissue microarray (TMA)-based approach to assess the frequency of R132H point mutations in formalin-fixed, paraffin-embedded tissue samples.
Materials and Methods : A total of 195 gliomas (30 pilocytic astrocytoma (PA), 45 diffuse astrocytoma [DA], 75 glioblastoma multiforme [GBM], 25 oligodendroglioma [OLIG] and 20 ependymoma [EPEN]). A TMA of core size 1.0 mm was constructed using a semi-automatic tissue arrayer. Immunohistochemical staining for IDH1, p53 and EGFR proteins was performed by the labeled sterptavidin avidin biotin LSAB method.
Results : The frequency of mutant IDH1 detection by immunohistochemistry on formalin-fixed, paraffin-embedded tissue was 15.8% in 29/184 tumors found suitable for evaluation. DA, OLIG and GBM showed IDH1 expression in 17/40 (42.5%), 5/22 (22.7%) and 7/72 (9.7%) cases, respectively. Of all the GBMs, prim-GBM showed immunoexpression in 1/7 (1.5%) while sec-GBM showed IDH1 expression in 6/7 (85.7%). PA and EPEN did not react with anti-IDH1 antibody. DA and GBM showed positive correlation with p53, but IDH1 and EGFR coexpression was rare.
Conclusion : Monoclonal antibody to IDH1 (R132) is a useful and less-labor-intensive method to detect mutations in gliomas. IDH1 is a useful immunohistochemical marker to differentiate reactive gliosis from low-grade astrocytoma, has potential as an independent prognostic marker and also helps in distinguishing primary from secondary GBM. Its sensitivity and specificity need to be assessed by simultaneous sequencing and its validation on clinically annotated samples.

Keywords: Glioma, IDH1 gene, immunohistochemistry, tissue microarray, biomarker


How to cite this article:
Sipayya V, Sharma I, Sharma K C, Singh A. Immunohistochemical expression of IDH1 in gliomas: A tissue microarray-based approach. J Can Res Ther 2012;8:598-601

How to cite this URL:
Sipayya V, Sharma I, Sharma K C, Singh A. Immunohistochemical expression of IDH1 in gliomas: A tissue microarray-based approach. J Can Res Ther [serial online] 2012 [cited 2019 Sep 20];8:598-601. Available from: http://www.cancerjournal.net/text.asp?2012/8/4/598/106567


 > Introduction Top


Gliomas arise from the glial cells, and constitute about half of all primary intracranial tumors. [1] Depending on the cell of origin, gliomas are mainly of three types: astrocytoma, oligodendroglioma (OLIG) and ependymoma (EPEN). Malignant gliomas are the most frequent and lethal cancers originating in the central nervous system. These include astrocytoma, OLIG and EPEN. The most frequent and biologically aggressive subtype is glioblastoma (GBM). Historically, GBM have been categorized into two groups - primary and secondary, on the basis of their clinical presentation. [2] Secondary GBM (sec-GBM) are defined as tumors that have clinical, radiologic or histopathologic evidence of malignant progression from a preexisting lower-grade tumor, whereas primary GBMs (prim-GBM) have no such history and present de novo as advanced cancers at the time of diagnosis. [3]

The histopathologic findings of primary and secondary GBMs are indistinguishable, and their prognosis does not appear to be significantly different after adjustment for age. [4],[5] However, these variants of GBM differ significantly with respect to genetic alteration. EGFR amplification, MDM 2 amplification and PTEN mutations are typical of prim-GBM while TP53 mutations are more frequent in sec-GBM. [6] Substantial research has been focused on identification of gene alterations in GBM that might help to stratify GBM patients depending on prognosis and response to therapy.

We screened a total of 195 gliomas for isocitrate dehydrogenase (IDH1) mutations using a tissue microarray (TMA)-based approach and assessed the role of immunohistochemical expression of IDH1 protein in these tumors with respect to different histological types and correlated IDH1 immunoexpression with p53 and EGFR expression.


 > Materials and Methods Top


Records of patients reported as glioma from January 2005 to June 2009 were studied and their archival FFPE blocks were retrieved. As this was a retrospective study and only the archival formalin-fixed blocks were used for TMA construction, the institutional ethical committee clearance was waived. Fresh hematoxylin and eosin (H and E)-stained sections were cut and evaluated independently by two pathologists and a consensus in the diagnosis as well as its WHO grade achieved. A total of 195 biopsies were included in this study. These included 30 pilocytic astrocytoma (PA), 45 diffuse astrocytoma (DA), 75 GBM, 25 OLIG and 20 EPEN. Among GBMs, tumors were considered as sec-GBM only when there was a prior histopathologically confirmed evidence of a preceding glioma.

A TMA was constructed as described by Avninder et al.[7] and all the 195 paraffin blocks were marked for area best representative of the tumor matching them with corresponding H&E-stained slides. A semiautomatic tissue arrayer, Minicore from Alphelys SAS, France, was used to transfer the donor tissue cylinders of 1.0 mm core diameter into the recipient block. The TMA block was tempered overnight at 37°C and 4-μ-thick sections were taken on poly-prep-coated slides (Sigma-Aldrich, St Louis, MO, USA). A check H and E stain was performed on the first section of the TMA to assess the degree of core loss [Figure 1]. There was a loss of nine cores (4.6%) after TMA construction, and 184 cores were found to be suitable for immunohistochemical staining.
Figure 1: Photomicrograph of tissue microarray showing subarrays of different glioma types (H&E, x20)

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TMA sections were deparaffinized through xylene and rehydrated. Slides were then incubated in 0.3% H 2 O 2 in methanol for 30 min to block endogenous peroxidase. Antigen retrieval was performed using a microwave-based EZ retrieval system, Biogenex, Fremont, CA, USA, and incubation with primary antibodies was done at 4°C. Antibodies used were IDH1 R132H (clone H09, dilution 1:20; Dianova, Hamburg, Germany); p 53 (clone DO7, 1:200 dilution; Dako) and EGFR (clone H11, 1:50 dilution; Dako). The detection system was LSAB and incubation with diaminobenzidine provided the chromogenic substrate. The sections were washed and counterstained with hematoxylin. A core was suitable for evaluation if at least 50% of the tumor cells were retained in the core. We examined the association between the presence of IDH1 expression and p53 and EGFR positivity in these gliomas using Fishers exact test. P values were considered statistically significant when P<0.05. Representative immunohistochemical staining in tissue microarray cores are shown in [Figure 2].
Figure 2: Photomicrograph of tissue microarray cores showing isocitrate dehydrogenase 1 expression in diffuse astrocytoma (DA) (a), glioblastoma multiforme (GBM) (b), oligodendroglioma (c), p53 in GBM (d), EGFR in GBM (e) and p53 in DA (x100)

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 > Results Top


A summary of the results is given in [Table 1]. None of the PA or EPEN showed any immunostaining with IDH1 protein. However, 17/40 (42.5%) DA showed positive labeling with IDH1 and 27/40 (67.5%) showed mutant p53. This IDH1 expression was positively correlated with p53 expression (P=0.018). OLIG also showed positive immunoexpression of IDH1 in 22.5% of the cases but mutant p53 in only 41% of the cases (P=0.07). Among primary GBM, only 1/65 showed IDH1 immunopositivity whereas 6/7(85%) of sec-GBM showed expression of this protein. Prim-GBM showed p53 and EGFR positivity in 64.5% and 60%, respectively. In the 6/7 sec-GBM that showed mutant IDH1 expression, 5/7 also showed mutant p53, but in GBM, coexpression of IDH1 and EGFR was rarely seen. Also, sec-GBM, which showed IDH1 mutations more frequently in comparison with prim-GBM, occurred in younger patients (mean age 42.5 years) as compared with prim-GBM (mean age of 47.2 years). Follow-up and survival data of most these patients was not available as ours is a tertiary referral hospital with more than half of the patients coming from other towns and villages across India.
Table 1: Immunohistochemical expression of IDH1, p53 and EGFR in gliomas

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 > Discussion Top


The IDH1 gene, located on chromosome 2 q33, encodes for an enzyme IDH1, which is one of the components of the citric acid cycle. This enzyme catalyzes the oxidative decarboxylation of isocitrate to alfa-ketoglutarate using either NAD or NADP as cosubstrates, resulting in NADPH production, and is thought to play a role in the cellular control of oxidative damage. [8] Parson et al.[9] studied 22 GBM tumor samples using high-density oligonucleotide arrays and sequenced 20,661 protein-coding genes. Their study revealed for the first time IDH1 as an unexpected target of genetic alteration in patients with GBM. This somatic mutation of IDH1 gene at codon 132 was reportedly also seen in prostate carcinoma and acute myeloid leukemia. [10],[11] Subsequent to this report, many researchers studied the role of IDH1 in brain tumors. Balss et al.[12] studied 685 brain tumors and found 221 somatic IDH1 mutations with a frequency of 68% in DA, 69% in OLIG and 88% in secondary GBM. Other tumors like prim-GBM, PA, EPEN, medulloblastomas and schwannomas showed absence of IDH1 mutations. The very high frequency of IDH1 mutations in DA and OLIG suggests a role in early tumor development. Ichimura et al.[13] screened exon 4 of gene IDH1 in 596 primary intracranial tumours of all major types, and found codon132 mutations in 54% of DA and 65% of OLIG but in only 6% of GBM (3% of prim-GBM and 50% of sec-GBM). No mutation in any other tumor type was seen. Nobusawa et al.[14] studied 407 GBMs to assess whether IDH1 mutation allows a reliable discrimination between prim-GBM and sec-GBM, and found that IDH1 mutations were detected in 36/407 (8.8%) of GBM and that the GBM patients with IDH1 mutations were younger and were associated with a longer survival. Capper et al.[15] used an antibody specific for IDH1 R132H mutation in 345 primary brain tumors, and demonstrated IDH1 immunoexpression in 44/53 (83%) DA, 9/10 (90%) OLIG, 0/21 PA, 5/7 (71%) sec-GBM and 2/56 (3.6%) prim-GBM. They concluded that immunohistochemistry was sensitive and specific in detecting IDH1 R132H mutation. Yan et al.[16] performed sequence analysis of IDH1 in 959 tumors, and the frequency of mutations at the R132 residue of IDH1 in DA, OLIG, prim GBM and sec-GBM was reported to be 83.3%, 80.3%, 4.8% and 84.6%, respectively. PA and EPEN did not show any mutation.

Watanabe et al.[17] assessed IDH1 mutation in 321 gliomas and detected a total of 130 IDH1 mutations. IDH mutations was frequent in DA (88%), sec-GBM (82%) and OLIG (79%), and IDH mutations copresent with TP53 mutation showed that there were no cases in which a IDH1 mutation occurred after the acquisition of TP53 mutation in DA or loss of 1 p/19 q in OLIG, suggesting that IDH1 mutations are early events in gliomagenesis. More recently, in a study published from India, Jha et al.[18] performed a mutational analysis on 100 gliomas of various grades and subtypes, and reported that IDH1 mutations occur in a large proportion of Indian glioma patients and that there was a significant positive correlation between IDH1 and p53 mutation, but no correlation of IDH1 mutation with EGFR amplification.

The knowledge of IDH1 status in gliomas is of both diagnostic as well as clinically prognostic relevance. On the diagnostic side, Camelo-Piragua et al.[19] showed that 9/21(42.9%) DA showed IDH1 mutation as compared with no mutation in 20 reactive gliosis biopsies. This demonstrates the utility of IDH1-immunohistochemistry in distinguishing low-grade astrocytoma from reactive astrocytosis, especially on small and diagnostically challenging biopsies. IDH1 mutations are also commonly seen in oligodendroglial tumors as in astrocyotmas. IDH1 status assists in separating oligodendroglial tumors from neurocytomas, clear cell EPENs or dysembryoplastic neuroepithelial tumor.

On the prognostic front, IDH1 mutations have been proposed to have an independent prognostic significance than the WHO diagnosis within a set of anaplastic astrocytomas (AA) and GBM, the order of outcome in terms of overall survival from most favorable to least favorable being AA with IDH1 mutation; GBM with mutation; AA without mutation and GBM without mutation. [20],[21],[22] IDH1 mutations are assessed by direct gene sequencing or immunohistochemistry. It is of paramount importance that no contamination from adjacent brain tissue, microglial cells or endothelial cells occurs as it will wrongly alter the results. Imunohistochemical detection of IDH1 mutation at codon 132 in trained hands gives 93% detection rate, [22] and is particularly useful in detecting single infiltrating tumor cells.

To conclude, immunohistochemical detection of IDH1 mutation using mutation-specific anti-IDH1 R132 mouse monoclonal antibody in FFPE tissue is a rapid and practical method of screening mutated IDH1 gene in gliomas. IDH1 mutation status segregates AA and GBM into clinically meaningful and prognostically distinct subgroups. IDH1 mutations are an independent prognostic factor for favorable prognosis and are a highly selective marker for sec-GBM, which complements clinical criteria for distinguishing pri- from sec-GBM. Given the diagnostic and prognostic implications of IDH1 mutations and potential for new therapies, testing for IDH1 mutations can enhance the accuracy of glioma diagnosis and treatment.


 > Acknowledgement Top


The authors wish to thank Dianova, Gmbh, Germany for providing a free sample of the IDH1 antibody at The 17 th International Congress of Neuropathology held at Salzburg, Austria in September, 2010

 
 > References Top

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9.Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al. An integrated genomic analysis of human glioblastoma multiforme. Science 2008;321:1807-12.  Back to cited text no. 9
    
10.Kang MR, Kim MS, Oh JE, Kim YR, Song SY, Seo SI, et al. Mutational analysis of IDH1 codon 132 in glioblastomas and other common cancers. Int J Cancer 2009;125:353-5.  Back to cited text no. 10
    
11.Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009;361:1058-66.  Back to cited text no. 11
    
12.Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol 2008;116:597-602.  Back to cited text no. 12
    
13.Ichimura K, Pearson DM, Kocialkowski S, Bäcklund LM, Chan R, Jones DT, et al. IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas. Neuro Oncol 2009;11:341-7.  Back to cited text no. 13
    
14.Nobusawa S, Watanabe T, Kleihues P, Ohgaki H. IDH1 mutations as molecular signature and predictive factor of secondary glioblastomas. Clin Cancer Res 2009;15:6002-7.  Back to cited text no. 14
    
15.Capper D, Weissert S, Balss J, Habel A, Meyer J, Jäger D, et al. Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors. Brain Pathol 2010;20:245-54.  Back to cited text no. 15
    
16.Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009;360:765-73.  Back to cited text no. 16
    
17.Watanabe T, Nobusawa S, Kleihues P, Ohgaki H. IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am J Pathol 2009;174:1149-53.  Back to cited text no. 17
    
18.Jha P, Suri V, Sharma V, Singh G, Sharma MC, Pathak P, et al. IDH1 mutations in gliomas : First series from a tertiary care centre in India with comprehensive review of literature. Exp Mol Pathol 2011;91:385-93.  Back to cited text no. 18
    
19.Camelo-Piragua S, Jansen M, Ganguly A, Kim JC, Louis DN, Nutt CL. Mutant IDH1-specific immunohistochemistry distinguishes diffuse astrocytoma from astrocytosis. Acta Neuropathol 2010;119:509-11.  Back to cited text no. 19
    
20.Weller M, Felsberg J, Hartmann C, Berger H, Steinbach JP, Schramm J, et al. Molecular predictors of progression-free and overall survival in patients with newly diagnosed glioblastoma : A prospective translational study of the German Glioma Network. J Clin Oncol 2009;27:5743-50.  Back to cited text no. 20
    
21.Hartmann C, Hentschel B, Wick W, Capper D, Felsberg J, Simon M, et al. Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1 mutation status accounts for the unfavorable prognostic effect of higher age : Implications for classification of gliomas. Acta Neuropathol 2010;120:707-18.  Back to cited text no. 21
    
22.von Deimling A, Korshunov A, Hartmann C. The next generation of glioma biomarkers : MGMT methylation, BRAF fusions and IDH1 mutations. Brain Pathol 2011;21:74-87.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]


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