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

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An Erratum to this article was published on 14 February 2017

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.

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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.).

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Authors and Affiliations

  1. Department of Neurosurgery and Neuro-Oncology, National Cancer Center Hospital, Tokyo, Japan

    Hideyuki Arita, Yoshitaka Narita, Yuko Matsushita, Yasuji Miyakita, Makoto Ohno & Soichiro Shibui

  2. Division of Brain Tumor Translational Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Hideyuki Arita, Shintaro Fukushima, Hirokazu Takami & Koichi Ichimura

  3. Department of Pathology and Clinical Laboratories, National Cancer Center Hospital, Tokyo, Japan

    Akihiko Yoshida

  4. Department of Neurosurgery, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Hirokazu Takami

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  1. Hideyuki Arita
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  2. Yoshitaka Narita
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Correspondence to Koichi Ichimura.

Additional information

An erratum to this article is available at http://dx.doi.org/10.1007/s10014-017-0277-9.

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Supplementary material 1 (XLSX 21 kb)

10014_2014_186_MOESM2_ESM.pptx

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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Arita, H., Narita, Y., Matsushita, Y. et al. Development of a robust and sensitive pyrosequencing assay for the detection of IDH1/2 mutations in gliomas. Brain Tumor Pathol 32, 22–30 (2015). https://doi.org/10.1007/s10014-014-0186-0

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  • DOI: https://doi.org/10.1007/s10014-014-0186-0

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