Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples.
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.
Yoshimoto
Koji
K
Departments of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1732, USA.
Dang
Julie
J
Zhu
Shaojun
S
Nathanson
David
D
Huang
Tiffany
T
Dumont
Rebecca
R
Seligson
David B
DB
Yong
William H
WH
Xiong
Zhenggang
Z
Rao
Nagesh
N
Winther
Henrik
H
Chakravarti
Arnab
A
Bigner
Darell D
DD
Mellinghoff
Ingo K
IK
Horvath
Steve
S
Cavenee
Webster K
WK
Cloughesy
Timothy F
TF
Mischel
Paul S
PS
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
C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors