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Development of a Real-time RT-PCR Assay for Detecting EGFRvIII in Glioblastoma Samples

Authors' Affiliations: Departments of ¹ Pathology and Laboratory Medicine, ² Molecular and Medical Pharmacology, ³ Human Genetics, ⁴ Biostatistics, and ⁵ Neurology, ⁶ Jonsson Comprehensive Cancer Center, and ⁷ Henry E. Singleton Brain Tumor Program, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California; ⁸ DAKO A/S, Glostrup, Denmark; ⁹ Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts; ¹⁰ Department of Pathology and The Preston Robert Tisch Brain Tumor Center at Duke, Durham, North Carolina; and ¹¹ Ludwig Institute for Cancer Research at University of California, La Jolla, San Diego, California

Requests for reprints: Paul S. Mischel, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte, Los Angeles, CA 90095-1732. Phone: 310-825-0377; Fax: 310-206-8290; E-mail: pmischel{at}mednet.ucla.edu.

Purpose: 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.

Experimental Design: 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).

Conclusion: 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.