title: A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma.
abstract: BACKGROUND
  Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.
  
  
  RESULTS
  The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results.
  
  
  CONCLUSIONS
  The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Catteau
authors.forename: Hélène
authors.initials: H
authors.lastname: Girardi
authors.forename: Florence
authors.initials: F
authors.lastname: Monville
authors.forename: Cécile
authors.initials: C
authors.lastname: Poggionovo
authors.forename: Sabrina
authors.initials: S
authors.lastname: Carpentier
authors.forename: Véronique
authors.initials: V
authors.lastname: Frayssinet
authors.forename: Jesse
authors.initials: J
authors.lastname: Voss
authors.forename: Robert
authors.initials: R
authors.lastname: Jenkins
authors.forename: Blandine
authors.initials: B
authors.lastname: Boisselier
authors.forename: Karima
authors.initials: K
authors.lastname: Mokhtari
authors.forename: Marc
authors.initials: M
authors.lastname: Sanson
authors.affiliation: QIAGEN Marseille, Av, de Luminy, Marseille, France. helene.peyro-saint-paul@qiagen.com.
authors.forename: Hélène
authors.initials: H
authors.lastname: Peyro-Saint-Paul
authors.forename: Caterina
authors.initials: C
authors.lastname: Giannini
mesh_terms: D000293:Adolescent; D001932:Brain Neoplasms / Q000175:diagnosis* / Q000235:genetics; D002648:Child; D066264:Datasets as Topic; D005260:Female; D005910:Glioma / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D008297:Male; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human