IDH1 mutation detection by droplet digital PCR in glioma

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.

Keywords: droplet digital PCR (ddPCR); glioma; isocitrate dehydrogenase (IDH); quantitative real-time PCR (qRT-PCR); sensitivity.

Figure 1. Detection profiles of the IDH1(R132H) mutation by ddPCR, qRT-PCR, and Sanger sequencing

A and B. The representative 1-D and 2-D plots of the ddPCR amplification profile of a IDH1 wild-type and a IDH1(R132H) mutant sample. The pink line in the left two rows indicates the threshold, and the orange dots in the upper-right quadrant of the right row indicate the mutant signal. C and D. The amplification curve of qRT-PCR of a IDH1 wild-type and a IDH1(R132H) mutant sample. The red line denotes the wild-type (WT-FAM) sample, and the blue line denotes the mutant (MT-VIC). E and F. The results of Sanger sequencing of the wild-type and IDH1(R132H) mutant samples (CGT > CAT).

Figure 2. The amplification results of a false-positive sample by ddPCR

A and B. The 1-D plots of positive channel 1-WT-FAM and false-positive channel 2-MT-VIC. C. The 2-D plot of WT-FAM and MT-VIC from a false-positive sample.

Figure 3. The detection limitations of ddPCR and qRT-PCR

A. The amplification results of a serially diluted sample derived from ddPCR and qRT-PCR. The number of dots above the threshold decreased following the dilution of the sample (the left three columns show the amplification of WT-FAM and MT-VIC and the 2-D plots with 2 channels). The right column is the amplification curve of qRT-PCR. B. The number of wild-type and mutant droplets detected in each diluted template. Blue, wild-type; green, mutant. C. The amplification curves of the mutant allele in the serially diluted template according to qRT-PCR.

Figure 4. IDH1 mutation is a positive prognostic marker for low-grade glioma patients

IDH1(R132H) mutation correlated with better overall survival in low-grade glioma patients in our cohort.