IDH mutation detection in formalin-fixed paraffin-embedded gliomas using multiplex PCR and single-base extension

Abstract

Isocitrate dehydrogenase (IDH) genes are mutated in a significant portion of gliomas, myeloid leukemias and chondroid neoplasms. In gliomas, IDH mutations are prognostic, as those tumors with the mutation are associated with a proneural subclass and have longer survival compared with those without the mutation. We developed a simple, PCR-based SNaPshot® assay (Life Technologies, Carlsbad, CA, USA) to detect IDH1/2 mutations. This protocol combines a single, multiplexed PCR reaction using gene specific primers followed by a single, multiplexed SNaPshot reaction and detection by capillary electrophoresis. In a blinded study of 32 paraffin-embedded glioma specimens previously screened for IDH mutations by a PCR/direct sequencing method, concordance of our IDH SNaPshot test with sequencing was 100%. We performed the assay on an additional 57 specimens submitted for diagnostic IDH mutation evaluation. Data analysis was much faster and easier to perform than analysis of the sequencing data, and results could be obtained in 1 day from DNA extraction to analysis. Furthermore, we could readily identify a mixture of 5% mutant allele vs. 95% wild-type allele in our SNaPshot assay, in comparison to approximately 20% mutant allele in our PCR-sequencing assay. Our assay represents a fast, sensitive, straightforward method of reliably detecting common mutations of IDH genes in glial neoplasms, or other tumors.

Figure 1

Representative electropherograms of IDH1 R132 mutant SNaPshot products. Panel A. Clinical sample with no isocitrate dehydrogenase (IDH) mutations. Panel B. Patient sample containing the IDH1 R132H mutation. Panel C. Patient sample showing the R132S mutation. Panel D. Control panel showing the common IDH1 and IDH2 mutant and wild‐type peaks.

Figure 2

Representative electropherograms of IDH2 R172 mutant SNaPshot products. Panel A. Clinical sample with no isocitrate dehydrogenase (IDH) mutations. Panel B. Patient sample containing the IDH2 R172K mutation. Panel C. Sequencing data (forward and reverse) showing heterozygosity of the IDH2 R172K mutation in the same patient specimen as above.

Figure 3

Comparison of sequencing and SNaPshot assay analytical sensitivity. Panel A. 60% R132H mutant(Mut):40% wild type(WT); Panel B. 40% Mut:60% WT; Panel C. 20% Mut:80% WT; Panel D. 10% Mut:90% WT. Arrow indicates position of the R132H mutant peak.

Figure 4

Titration of SNaPshot assay to assess assay maximum analytical sensitivity. Panel A. 20% R132H mutant, 80% wild‐type PCR product. Panel B. 10% mutant, 90% wild type. Panel C. 5% mutant, 95% wild type. Panel D. 2.5% mutant, 97.5% wild type. Panel E. 1% mutant, 99% wild type. Panel F. 100% wild type.