title: Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples.
abstract: OBJECTIVE
  Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge.
  
  
  METHODS
  We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization.
  
  
  RESULTS
  The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00).
  
  
  CONCLUSIONS
  This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.
authors.affiliation: Departments of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1732, USA.
authors.forename: Koji
authors.initials: K
authors.lastname: Yoshimoto
authors.forename: Julie
authors.initials: J
authors.lastname: Dang
authors.forename: Shaojun
authors.initials: S
authors.lastname: Zhu
authors.forename: David
authors.initials: D
authors.lastname: Nathanson
authors.forename: Tiffany
authors.initials: T
authors.lastname: Huang
authors.forename: Rebecca
authors.initials: R
authors.lastname: Dumont
authors.forename: David B
authors.initials: DB
authors.lastname: Seligson
authors.forename: William H
authors.initials: WH
authors.lastname: Yong
authors.forename: Zhenggang
authors.initials: Z
authors.lastname: Xiong
authors.forename: Nagesh
authors.initials: N
authors.lastname: Rao
authors.forename: Henrik
authors.initials: H
authors.lastname: Winther
authors.forename: Arnab
authors.initials: A
authors.lastname: Chakravarti
authors.forename: Darell D
authors.initials: DD
authors.lastname: Bigner
authors.forename: Ingo K
authors.initials: IK
authors.lastname: Mellinghoff
authors.forename: Steve
authors.initials: S
authors.lastname: Horvath
authors.forename: Webster K
authors.initials: WK
authors.lastname: Cavenee
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Cloughesy
authors.forename: Paul S
authors.initials: PS
authors.lastname: Mischel
mesh_terms: D066246:ErbB Receptors / Q000032:analysis*; D005909:Glioblastoma / Q000737:chemistry*; D006801:Humans; D016612:Paraffin Embedding; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors