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Brain Tumor Pathology

, Volume 32, Issue 1, pp 22–30 | Cite as

Development of a robust and sensitive pyrosequencing assay for the detection of IDH1/2 mutations in gliomas

  • Hideyuki Arita
  • Yoshitaka Narita
  • Yuko Matsushita
  • Shintaro Fukushima
  • Akihiko Yoshida
  • Hirokazu Takami
  • Yasuji Miyakita
  • Makoto Ohno
  • Soichiro Shibui
  • Koichi Ichimura
Original Article

Abstract

Assessment of the mutational status of the isocitrate dehydrogenase 1/2 (IDH1/2) gene has become an integral part of the standard diagnostic procedure and, therefore, needs to be accurate. This may, however, be compromised by various factors including the method of analysis and a low tumor cell content. We have developed a rapid, sensitive and robust assay to detect all types of mutation in either IDH1 or IDH2 using pyrosequencing. The efficacy of detecting mutation was evaluated using a panel of control plasmids representing all the different types of IDH1/2 mutation and a set of 160 tumor specimens. The sensitivity of the assays was examined by a serial dilution analysis performed on samples containing various ratios of wild-type and mutant alleles. The pyrosequencing assay detected as little as 5 % of mutant alleles for most mutation types, while conventional Sanger sequencing required the presence of at least 20 % of mutant alleles for identifying mutations. The pyrosequencing assay detected IDH1/2 mutations in three samples which were missed by Sanger sequencing due to their low tumor cell contents. Our assay is particularly useful for the analysis of a large number of specimens as in a retrospective clinical study for example.

Keywords

Glioma IDH1 IDH2 Pyrosequencing Mutation detection 

Notes

Acknowledgments

The authors thank Dr. Sylvia Kocialkowski for critical reading of the manuscript. This work was supported by JSPS KAKENHI Grant Numbers 24659650 (H. A.), 25462283 (K. I.) and by the National Cancer Center Research and Development Fund 23-A-50 (K. I.).

Supplementary material

10014_2014_186_MOESM1_ESM.xlsx (22 kb)
Supplementary material 1 (XLSX 21 kb)
10014_2014_186_MOESM2_ESM.pptx (106 kb)
Supplementary Figure 1 The pyrograms of IDH1 testing for matched FFPE and frozen samples in representative cases. The peaks indicating R132H mutations (arrow) appeared clearly in the pyrograms for FFPE, and the results were consistent with those for frozen samples (PPTX 106 kb)

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Copyright information

© The Japan Society of Brain Tumor Pathology 2014

Authors and Affiliations

  • Hideyuki Arita
    • 1
    • 2
  • Yoshitaka Narita
    • 1
  • Yuko Matsushita
    • 1
  • Shintaro Fukushima
    • 2
  • Akihiko Yoshida
    • 3
  • Hirokazu Takami
    • 2
    • 4
  • Yasuji Miyakita
    • 1
  • Makoto Ohno
    • 1
  • Soichiro Shibui
    • 1
  • Koichi Ichimura
    • 2
  1. 1.Department of Neurosurgery and Neuro-OncologyNational Cancer Center HospitalTokyoJapan
  2. 2.Division of Brain Tumor Translational ResearchNational Cancer Center Research InstituteTokyoJapan
  3. 3.Department of Pathology and Clinical LaboratoriesNational Cancer Center HospitalTokyoJapan
  4. 4.Department of NeurosurgeryUniversity of TokyoTokyoJapan

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