Abstract
Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.
Highlights
Glioma is a common adult central nervous system tumor
We evaluated the sensitivity and specificity of droplet digital PCR (ddPCR) and quantitative real-time PCR (qRT-PCR) compared with the current standard method, Sanger direct sequencing
Pilot experiments of ddPCR were performed to establish the assay conditions for IDH1(R132H) detection
Summary
Glioblastoma (GBM), Grade IV glioma, is the most lethal brain tumor (only 12 to 14 months after diagnosis). IDH1 and IDH2 catalyze the same reaction outside the context of the citric acid cycle and use NADP+. Wild-type IDH1 converts isocitrate to α-ketoglutarate (a potential oncometabolite), whereas the mutant IDH1 yields a neomorphic enzymatic function and catalyzes α-ketoglutarate into α-hydroxyglutarate, which is an www.impactjournals.com/oncotarget oncometabolite that is related to genomic hypertension, genetic instability, and malignant transformation [4]. The IDH1 mutation is one of the most common and earliest genetic alterations in glioma and is an effective diagnostic and predictive marker in glioma patients. Jose et al [5] investigated The Cancer Genome Atlas (TCGA) data and found that the level of pyruvate carboxylase was higher in human gliomas containing the IDH1 mutation than in those with wild-type IDH1. In addition to the important role in glioma, IDH mutations were found in myeloid neoplasia, peripheral T-cell lymphoma, chondrosarcoma, chonangiocarcinoma, prostate cancer, and other cancers [7, 8]
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