A novel COLD-PCR/FMCA assay enhances the detection of low-abundance IDH1 mutations in gliomas.
Point mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in many gliomas. The detection of IDH1 mutations becomes challenging on suboptimal glioma biopsies when a limited number of tumor cells is available for analysis. Coamplification at lower denaturing-polymerase chain reaction (COLD-PCR) is a PCR technique that deliberately lowers the denaturing cycle temperature to selectively favor amplification of mutant alleles, allowing for the sensitive detection of low-abundance mutations. We developed a novel COLD-PCR assay on the LightCycler platform (Roche, Applied Science, Indianapolis, IN), using post-PCR fluorescent melting curve analysis (FMCA) for the detection of mutant IDH1 with a detection limit of 1%. Thirty-five WHO grade I to IV gliomas and 9 non-neoplastic brain and spinal cord biopsies were analyzed with this technique and the results were compared with the conventional real-time PCR and the Sanger sequencing analysis. COLD-PCR/FMCA was able to detect the most common IDH1 R132H mutation and rare mutation types including R132H, R132C, R132L, R132S, and R132G mutations. Twenty-five glioma cases were positive for IDH1 mutations by COLD-PCR/FMCA, and 23 gliomas were positive by the conventional real-time PCR and Sanger sequencing. A pilocytic astrocytoma (PA I) and a glioblastoma multiforme (GBM IV) showed low-abundance IDH1 mutations detected by COLD-PCR/FMCA. The remaining 10 glioma and 9 non-neoplastic samples were negative by all the 3 methods. In summary, we report a novel COLD-PCR/FMCA method that provides rapid and sensitive detection of IDH1 mutations in formalin-fixed paraffin-embedded tissue and can be used in the clinical setting to assess the small brain biopsies.
Division of Molecular Anatomic Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Brendan
B
Pang
Mary B
MB
Durso
Ronald L
RL
Hamilton
Marina N
MN
Nikiforova
D001706:Biopsy; D005910:Glioma / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D020125:Mutation, Missense; D009691:Nucleic Acid Denaturation; D057089:Pathology, Molecular / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D044366:Transition Temperature
D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human