Detection of isocitrate dehydrogenase 1 mutation R132H in myelodysplastic syndrome by mutation-specific antibody and direct sequencing

doi:10.1016/j.leukres.2010.02.014

Leukemia Research

Volume 34, Issue 8, August 2010, Pages 1091-1093

https://doi.org/10.1016/j.leukres.2010.02.014 Get rights and content

Abstract

Sequencing of the acute myeloid leukemia genome revealed somatic mutations in isocitrate dehydrogenase-1. Acute myeloid leukemia frequently develops from myelodysplastic syndrome. In order to test whether myelodysplastic syndrome also carries isocitrate dehydrogenase-1 mutations, we stained a series of bone marrow samples from patients with myelodysplastic syndrome using an antibody specific for the R132H mutation. Three out of 71 patients exhibited antibody binding to myeloid precursor cells. The presence of the R132H mutation was confirmed by DNA sequencing. We demonstrated that isocitrate dehydrogenase-1 mutations occur in myelodysplasia preceding acute myeloid leukemia and that the R132H alteration can be detected by immunohistochemistry.

Introduction

Somatic mutations in the gene encoding cytosolic NADP+-dependent isocitrate dehydrogenase 1 (IDH1) are very frequent in human glioma, but extremely rare in other solid tumors [1], [2], [3]. R132H mutation dominantly inhibits IDH1 activity and induces hypoxia-inducible factor 1 (HIF1) [4]. Thus IDH1 may function as a tumor suppressor that might contribute to tumorigenesis in part through induction of the HIF1 pathway [4].

Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasia characterized by heterogeneous genomic aberrations of myeloid precursor cells resulting in maturation arrest and bone marrow (BM) infiltration by blasts. In about 45–50% of AML no chromosomal abnormalities but hidden genomic alterations and somatic mutations can be detected. Recently IDH1 R132 mutations had been found in 10% of de novo AML [5] and mutated status was significantly associated with normal karyotype suggesting that these mutations are not random and are probably important for the pathogenesis of AML.

Myelodysplastic syndrome (MDS) is another clonal bone marrow disorder that shares with AML several pathogenetic mechanisms including common chromosomal aberrations and somatic mutations. Furthermore, MDS is characterized by presence of neoplastic sub-clones of myeloid precursor cells that accumulate genomic alterations and finally progress to AML with variable frequency. At the initial stages of MDS, the clonal stem cells usually represent a minority of BM cells. It is therefore very difficult to demonstrate the neoplastic nature of MDS, and the pathogenesis of MDS in general still remains obscure.

The aim of this study was to investigate a series of MDS cases for IDH1 R132H mutation using immunohistochemistry with a recently developed R132H mutation-specific monoclonal antibody [6].

Section snippets

Tissue specimens

BM-biopsies were randomly retrieved from the archives of the Pathology Department of Institute of Pathology Heidelberg (Heidelberg, Germany). The routine diagnosis was established according to World Health Organization (WHO) criteria on formalin-fixed, paraffin-embedded tissue specimens and peripheral blood/bone marrow smears. All diagnoses were reviewed and confirmed by two of the authors (MA and TL).

Immunohistochemistry

IDH1 R132H immunostaining was performed using a Ventana BenchMark XT immuno stainer (Ventana

Results and discussion

Immunohistochemical staining of 71 BM-biopsies of MDS patients using the IDH1 R132H mutation-specific antibody revealed three cases with positive hematopoietic cells. The mutated IDH1 was detectable in the cytoplasm in of granulocyte precursor cells and in few dysplastic megakaryocytes (Fig. 1A–C). This result suggests that mutant IDH1 occurs early in the course of disease and initially only affects few myeloid precursor cells that populate bone marrow together with the residual non-neoplastic

Conflict of interest statement

The authors report no potential conflicts of interest.

Acknowledgements

The authors would like to thank Charlotte Wahrendorf, Diana Jäger and Jochen Meyer for technical assistance. This work was supported by grants from the Bundesministerium fuer Bildung und Forschung (No. BMBF01ES0730 and No. BMBF01GS0883).

Contributions: AvD was the principal investigator and takes primary responsibility for the paper. HZ, DC and AvD developed the IDH antibody. TL and MA selected cases and analyzed data. DC, CH, AvD and MA evaluated the laboratory work. AvD and MA coordinated

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IDH1 mutations at residue p.R132 (IDH1(R132)) occur frequently in high-grade gliomas but not in other solid tumors
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Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas
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There are more references available in the full text version of this article.

Cited by (16)

A gated material as immunosensor for in-tissue detection of IDH1-R132H mutation in gliomas
2021, Sensors and Actuators, B: Chemical
Citation Excerpt :
The most habitual mutation detected was R132H (89.7 %), but other mutations have also been reported, for example R132C (2.8 %), R132 K (2.8 %), R132S (1.4 %) and R132 G (1.1 %) [9]. To date, this potential biomarker has been detected by routine clinical analysis such as direct sequencing [10,11], western-blot [12] and immunohistochemistry in tumour tissue using different anti-IDH1 (R132 H) antibodies [13–15]. Although some of them present high accuracy, these techniques are time consuming and require complex and expensive equipment [16].
A nanodevice consisted on nanoporous anodic alumina (NAA) supports functionalized with specific and selective antibody-based gatekeepers for the detection of IDH1-R132H mutant enzyme is here reported. Molecular profile and tissue mutations of the tumours (such as IDH1/IDH2 mutations in gliomas) are a great source of information that already make a difference in terms of prognosis and prediction of response to combined therapy. However, standardized methodologies to determine this mutation are time-consuming and cannot provide information before or during surgical intervention, which significantly limits their utility in terms of intraoperative decision-making. To solve this limitation, our sensing system, in the presence of the target IDH1-R132H mutant enzyme triggers the delivery of a fluorescent reporter from the antibody-capped NAA that is easily detected using a standard fluorimeter in less than 1 h. Good capabilities of the sensor in terms of sensitivity (limit of detection as low as 0.01 ng/mL) and selectivity are determined. The biosensor is validated in seventeen human glioma tissue samples, also analysed by standardized methodologies, showing significant differences between IDH1 wild-type and IDH1-R132H mutant gliomas (p < 0.01) with high sensitivity, specificity and accuracy (87.5/88.9/88.2) for IDH1-R132H mutational status classification in human glioma tissues. This new detection system might play an important role in surgical interventions, anticipating mutational status information in gliomas and supporting thus decision making and patient survival.
IDH mutations in acute myeloid leukemia
2012, Human Pathology
Acute myeloid leukemia is a heterogeneous group of diseases. Mutations of the isocitrate dehydrogenase (IDH) genes represent a novel class of point mutations in acute myeloid leukemia. These mutations prevent oxidative decarboxylation of isocitrate to α-ketoglutarate and confer novel enzymatic activity, facilitating the reduction of α-ketoglutarate to d-2-hydroxyglutarate, a putative oncometabolite. IDH1/IDH2 mutations are heterozygous, and their combined frequency is approximately 17% in unselected acute myeloid leukemia cases, 27% in cytogenetically normal acute myeloid leukemia cases, and up to 67% in acute myeloid leukemia cases with cuplike nuclei. These mutations are largely mutually exclusive. Despite many similarities of IDH1 and IDH2 mutations, it is possible that they represent distinct molecular or clinical subgroups of acute myeloid leukemia. All known mutations involve arginine (R), in codon 132 of IDH1 or codon 140 or 172 of IDH2. IDH1^R132 and IDH2^R140 mutations are frequently accompanied by normal cytogenetics and NPM1 mutation, whereas IDH2^R172 is frequently the only mutation detected in acute myeloid leukemia. There is increasing evidence that the prognostic impact of IDH1/2 mutations varies according to the specific mutation and also depends on the context of concurrent mutations of other genes. IDH1^R132 mutation may predict poor outcome in a subset of patients with molecular low-risk acute myeloid leukemia, whereas IDH2^R172 mutations confer a poor prognosis in patients with acute myeloid leukemia. Expression of IDH1/2 mutants induces an increase in global DNA hypermethylation and inhibits TET2-induced cytosine 5-hydroxymethylation, DNA demethylation. These data suggest that IDH1/2 mutations constitute a distinct mutational class in acute myeloid leukemia, which affects the epigenetic state, an important consideration for the development of therapeutic agents.
Isocitrate dehydrogenase mutation hot spots in acute lymphoblastic leukemia and oral cancer
2012, Kaohsiung Journal of Medical Sciences
Citation Excerpt :
Mutations of IDH impair the ability to catalyze the conversion of isocitrate to ɑ-ketoglutarate (ɑ-KG), which decreases conversion of ɑ-KG to the metabolite D2-hydroxyglutarate. Of the three IDH isoforms (IDH1, IDH2, and IDH3), two isoforms (IDH1 and IDH2) are associated with brain tumors [3–10], acute myeloid leukemia (AML) [11–15], myelodysplastic syndromes [16,17], prostate cancer [9], acute lymphoblastic leukemia (ALL) B [9], paraganglioma [18], thyroid cancer [19], and colorectal cancer [3,20]. Like tumor suppressors, heterozygous missense mutations of IDH1 residues act in a dominant negative fashion [18].
Isocitrate dehydrogenase (IDH) encodes a nicotinamide adenine dinucleotide phosphate+-dependent enzyme for oxidative decarboxylation of isocitrate and has an essential role in the tricarboxylic acid cycle. Mutations of IDH1 and IDH2 have been identified in patients with glioma, leukemia, and other cancers. However, the incidence of IDH mutations in acute myeloid leukemia in Taiwan is much lower than that reported in Western countries. The reason for the difference is unknown and its clinical implications remain unclear. Acute lymphoblastic leukemia (ALL) is a heterogenous hematopoietic malignancy. Oral squamous cell carcinoma (OSCC) results from chronic carcinogen exposures and is highly prevalent in trucking workers, especially in southern Taiwan. Subtypes of both diseases require specific treatments, and molecular markers for developing tailored treatments are limited. High-resolution melting (HRM) analysis is now a widely used methodology for rapid, accurate, and low-cost mutation scanning. In this study, 90 adults with OSC and 31 children with ALL were scanned by HRM analysis for IDH1 and IDH2 mutation hot spots. In ALL, the allele frequency was 3.23% in both IDH1 and IDH2. In OSCC, the allele frequency was 2.22% in IDH2. A synonymous mutation over pG313 (c.939A > G) of IDH2 was found in both pediatric ALL and adult OSCC. Therefore, we concluded that mutations of IDH are uncommon in ALL and OSCC and are apparently not a major consideration when selecting treatment modalities.
Rapid and reliable detection of IDH1 R132 mutations in acute myeloid leukemia using high-resolution melting curve analysis
2011, Clinical Biochemistry
Citation Excerpt :
Identification of such mutation might be useful to provide a biomarker for further stratifying patients and monitoring minimal residual disease in cytogenetically normal AML. Several methods have been established to detect IDH1 mutation, including direct DNA sequencing, PCR followed by restriction fragment length polymorphism (RFLP) gel electrophoresis, PCR-single strain conformation polymorphism (SSCP) electrophoresis, and R132H antibody detection [2–17]. Each of these methods has its own merits and limitations, including sensitivity, ease of use, and low throughput.
The mutations of isocitrate dehydrogenase 1 (IDH1) gene have been identified in a proportion of hematologic malignancies including acute myeloid leukemia (AML). The aim of the present study was to explore the reliability of the high-resolution melting analysis (HRMA) for the identification of IDH1 R132 mutations in AML.
We evaluated the sensitivity of HRMA in the detection of IDH1 R132 mutation and screened IDH1 mutations in 110 AML patients using HRMA. The results of HRMA were validated by direct DNA sequencing.
The reproducible sensitivity of HRMA was 5% for the detection of IDH1 R132 mutation, higher than 10% of direct DNA sequencing. Heterozygous IDH1 mutations were identified in 4 (3.6%) AML cases, which were R132H in 3 cases and R132S in 1 case confirmed by DNA sequencing.
The HRMA is a rapid, accurate, reliable, high-throughput method to screen IDH1 gene mutations.
IDH mutations in glioma and acute myeloid leukemia
2010, Trends in Molecular Medicine
Citation Excerpt :
For one, the AML case shares important similarities to GBM in that IDH mutation already exists in several related precursor diseases often antecedent to the development of AML, parallel to the case of low-grade gliomas that progress to become secondary GBM maintaining the IDH mutation throughout disease progression. In addition to primary AML, recurrent IDH1 were found in 5–10% of sAML, MDS and MPN [29,38,57]. MDS and MPN are also clonal proliferative blood disorders affecting the bone marrow and share many of the common cytogenetic and somatic alterations found in primary AML.
The systematic sequencing of glioblastoma multiforme (GBM) genomes has identified the recurrent mutation of IDH1, a gene encoding NADP⁺-dependent isocitrate dehydrogenase 1 (IDH1) that catalyzes the oxidative decarboxylation of isocitrate yielding α-ketoglutarate (α-KG). Subsequent studies have confirmed recurrent IDH1 and IDH2 mutations in up to 70% of low-grade glioma and secondary GBM, as well as in 10% of acute myeloid leukemia (AML) cases. The heterozygous somatic mutations at arginine R132 (IDH1) and at R140 or R172 (IDH2) in the enzyme active site confer a gain of function to the enzymes, which can both produce the metabolite 2-hydroxyglutarate. This review surveys the prevalence of IDH mutations in cancer and explores current mechanistic understanding of IDH mutations with implications for diagnostic and therapeutic development for the treatment of gliomas and AML.
Emerging Immunohistochemical Biomarkers for Myeloid Neoplasms
2023, Archives of Pathology and Laboratory Medicine

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Brief communicationDetection of isocitrate dehydrogenase 1 mutation R132H in myelodysplastic syndrome by mutation-specific antibody and direct sequencing

Abstract

Introduction

Section snippets

Tissue specimens

Immunohistochemistry

Results and discussion

Conflict of interest statement

Acknowledgements

IDH1 mutations at residue p.R132 (IDH1(R132)) occur frequently in high-grade gliomas but not in other solid tumors

Hum Mutat

Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas

Acta Neuropathol

An integrated genomic analysis of human glioblastoma multiforme

Science

Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha

Science

Brief communication
Detection of isocitrate dehydrogenase 1 mutation R132H in myelodysplastic syndrome by mutation-specific antibody and direct sequencing