Abstract
The current World Health Organization classification of diffuse astrocytic and oligodendroglial tumors requires the examination of isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations. Conventional analysis tools, including Sanger DNA sequencing or pyrosequencing, fail in detecting these variants of low frequency owing to their limited sensitivity. Digital polymerase chain reaction (dPCR) is a recently developed, highly sensitive, and precise quantitative rare variant assay. This study aimed to establish a robust limit of quantitation of the dPCR assay to detect a small fraction of IDH1 R132H mutation. The dPCR assays with serially diluted IDH1 R132H constructs detected 0.05% or more of mutant IDH1 R132H in samples containing mutant DNA. The measured target/total value of the experiments was proportional to the dilution factors and was almost equal to the actual frequencies of the mutant alleles. Based on the average target/total values, together with a twofold standard deviation of the normal DNA, a limit of quantitation of 0.25% was set to secure a safe margin to judge the mutation status of the IDH1 R132H dPCR assay. In clinical settings, detecting IDH1 R132H using dPCR assays can validate ambiguous immunohistochemistry results even when conventional DNA sequencing cannot detect the mutation and assure diagnostic quality.
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Acknowledgements
The authors thank Sachiko Miura, Chizu Kina, and Toshiko Sakaguchi for their technical assistance. The Department of Brain Disease Translational Research is an endowment department supported by an unrestricted grant from Idorsia Pharmaceuticals Japan Ltd. This work was supported in part by Grant-in-Aid for Young Scientists (B) and for Scientific Research (C) (KAKENHI) from the Japan Society for the Promotion of Science (17K15659 and 22K06951, K. Satomi).
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Japan Society for the Promotion of Science, 22K06951, Kaishi Satomi, 17K15659, Kaishi Satomi
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Satomi, K., Yoshida, A., Matsushita, Y. et al. Clinical application of a highly sensitive digital PCR assay to detect a small fraction of IDH1 R132H-mutant alleles in diffuse gliomas. Brain Tumor Pathol 39, 210–217 (2022). https://doi.org/10.1007/s10014-022-00442-5
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DOI: https://doi.org/10.1007/s10014-022-00442-5