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Rapid and sensitive assessment of the IDH1 and IDH2 mutation status in cerebral gliomas based on DNA pyrosequencing

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Abstract

Diffusely infiltrating cerebral gliomas frequently carry point mutations in codon 132 of the isocitrate dehydrogenase 1 (IDH1) gene or in codon 172 of the IDH2 gene, which are both clinically important as diagnostic and prognostic markers. Here, we report on a method that allows for the rapid detection of IDH1 and IDH2 mutations based on pyrosequencing. The method is applicable to routinely processed tissue specimens and provides quantitative mutation data within less than one working day. Due to its high sensitivity, the technique may also be used for the diagnostic assessment of IDH1 or IDH2 mutation in tissue samples with low tumor cell content, such as the infiltration zone of diffuse gliomas. Using pyrosequencing and/or conventional cycle sequencing of IDH1 and IDH2 in 262 gliomas, we confirm frequent mutations in diffuse astrocytic and oligodendroglial gliomas, corroborate a prognostic role for IDH1 mutation in primary glioblastomas and show that pleomorphic xanthoastrocytomas generally lack mutations in these genes.

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Authors and Affiliations

  1. Department of Neuropathology, Heinrich-Heine-University, Moorenstrasse 5, 40225, Düsseldorf, Germany

    Jörg Felsberg, Marietta Wolter, Heike Seul, Britta Friedensdorf & Guido Reifenberger

  2. Department of Neurosurgery, Heinrich-Heine-University, Düsseldorf, Germany

    Matthias Göppert & Michael C. Sabel

Authors
  1. Jörg Felsberg
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  2. Marietta Wolter
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  3. Heike Seul
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  4. Britta Friedensdorf
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  5. Matthias Göppert
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  6. Michael C. Sabel
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  7. Guido Reifenberger
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Corresponding author

Correspondence to Guido Reifenberger.

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Fig. S1

Location of the primer sequences used for IDH1 (A) and IDH2 (B) pyrosequencing. The PCR primer binding sites used in our analyses are labeled in grey (IDH1-For_Bio and IDH1-Rev for IDH1 amplification; IDH2-For and IDH2-Rev_Bio for IDH2 amplification). Location of the primers used for pyrosequencing are printed in blue and underlined (IDH1-PySeq and IDH2-PySeq, respectively). The commonly mutated codons 132 in IDH1 and 172 in IDH2 are printed in bold and labeled in yellow. In case of IDH1, the PCR primer binding sites used for amplification of a shorter amplicon of 72 bp with primers IDH1-For_Bio_short and IDH1-Rev_short are also indicated (red boxes). (DOC 19.5 kb)

Fig. S2

Comparison of IDH1 pyrosequencing results obtained on DNA extracted from frozen (cryo) or formalin-fixed paraffin-embedded (paraf) tissue samples of 10 different IDH1 mutant gliomas. Note that all mutations detected in high-molecular weight DNA extracted from frozen tumor tissue specimens were also detected in DNA samples obtained from paraffin material (values from corresponding sample pairs are indicated by the same color). Furthermore, the median percentages of mutant alleles detected by pyrosequencing (indicated by horizontal lines) were similar for both template sources, i.e., 39.5% (range: 18–43%) vs. 38.5% (range 30–46%). (TIFF 2.48 Mb)

Fig. S3

Sensitivity of IDH1 mutation detection by pyrosequencing. Shown are results of a dilution series using variable mixtures of IDH1 wild-type DNA and DNA from the tumor AA231, which carried a homozygous IDH1 mutation (c.395G>A; R132H) with a mutant allele percentage of 84% as determined by pyrosequencing. The ordinate indicates the mutant allele frequencies as detected by pyrosequencing. The abscissa shows the different concentrations of AA231 DNA in percent that were used as templates. The insert shows an enlarged part of the figure corresponding to the dilutions from 0 to 10% AA231 DNA. Note that pyrosequencing allows for a quantitative detection of mutant alleles and is already able to detect the IDH1 mutation when the template contained at least 4% of AA231 tumor DNA. To distinguish the presence of a mutation from background noise, a threshold of 5% mutant allele frequency (indicated by the horizontal line) is often used (4, 17) and recommended in the manual of the Pyromark Q24 machine (Qiagen). In our experiments, this 5% threshold was surpassed at a concentration of 8% of AA231 tumor DNA, corresponding to approximately 7% of mutant alleles. (TIFF 2.32 Mb)

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Felsberg, J., Wolter, M., Seul, H. et al. Rapid and sensitive assessment of the IDH1 and IDH2 mutation status in cerebral gliomas based on DNA pyrosequencing. Acta Neuropathol 119, 501–507 (2010). https://doi.org/10.1007/s00401-010-0647-4

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