IDH1 mutations in oligodendroglial tumors: comparative analysis of direct sequencing, pyrosequencing, immunohistochemistry, nested PCR and PNA-mediated clamping PCR - PubMed (original) (raw)

Comparative Study

IDH1 mutations in oligodendroglial tumors: comparative analysis of direct sequencing, pyrosequencing, immunohistochemistry, nested PCR and PNA-mediated clamping PCR

Dakeun Lee et al. Brain Pathol. 2013 May.

Abstract

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in a high proportion of glial tumors and have a significant prognostic impact. Although direct sequencing has been considered to be the gold-standard method to detect this mutation, the sensitivity of this technique has been questioned especially because specimens from glial tumors may contain large numbers of non-tumor cells. We screened 141 cases of oligodendroglial tumors for IDH1 mutations using peptide nucleic acid (PNA)-mediated clamping polymerase chain reaction (PCR) and compared the results with the results of direct sequencing, pyrosequencing, and immunohistochemistry (IHC). Nested PCR was only performed in cases having mutant IDH1 only discovered by clamping PCR. Using dilution experiments mixing IDH1 wild-type and mutant DNA samples, clamping PCR detected mutations in samples with a 1% tumor DNA composition. Using PNA clamping PCR, we detected 138 of 141 (97.9%) cases with mutant IDH1 in our series, which is significantly higher (P = 0.016; PNA clamping vs. direct sequencing) than those of direct sequencing (74.5%), pyrosequencing (75.2%) and IHC (75.9%). From our results, almost all oligodendroglial tumors have IDH1 mutations, and this suggests that IDH1 mutation is an early and common event especially in the development of oligodendroglial tumors.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1

The peptide nucleic acid (

PNA

) clamping system. The

PNA

oligomer was designed to bind to the bottom strand of the wild‐type sequence, spanning mutational hot spot of the isocytrate dehydrogenase 1 gene. The forward polymerase chain reaction (

PCR

) primer partially overlapped the

PNA

binding site. (A) A

PNA

DNA

hybrid with a perfect match prevents annealing of the

PCR

primer and amplification of wild‐type

DNA

. (B) A

PNA

DNA

hybrid with a single‐base pair mismatch does not suppress annealing of the

PCR

primer or amplification of mutant alleles.

Figure 2

The flowchart and time schedule of the peptide nucleic acid (

PNA

) clamping method used for the detection of IDH1 mutations in routinely processed glioma tissue specimens. Note that the total time required from cutting paraffin sections to obtaining sequence results amounts to approximately 3 h.

Figure 3

Results of dilution experiments mixing IDH1 wild‐type (from

87‐

human glioblastoma‐astrocytoma cell line) and mutant (from a mutant clone)

DNA

samples in different proportions. These results indicate that the

PNA

clamping method is able to detect IDH1 mutations with a mutant allele frequency of 1% or more by our standards (Δ

t1 > 2.0).

Figure 4

Kaplan‐Meier survival curves for overall survival in patients with (i) anaplastic oligodendroglioma (

OIII

); and (ii) anaplastic oligoastrocytoma (

OAIII

) according to the Δ

t1 values from clamping

PCR

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IDH1 mutations in oligodendroglial tumors: comparative analysis of direct sequencing, pyrosequencing, immunohistochemistry, nested PCR and PNA-mediated clamping PCR - PubMed (original) (raw)