Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Assay for Detecting Epidermal Growth Factor Receptor Variant-III (EGFRvIII) in Glioblastomas
A Raghunathan, G Sharma, L Whiteley, JA Gutierrez, D Chitale. Henry Ford Hospital, Detroit, MI
Background: The EGFRvIII is an oncogene generated by an in-frame genomic deletion that defines a prognostically distinct subgroup of glioblastomas associated with favorable response to tyrosine kinase inhibitor therapy. Detection of this mutation in clinical samples, therefore, is warranted. This is challenged by the unavailability of frozen tumor tissues, especially in a community hospital setting, and lack of a reliable EGFRvIII antibody for immunohistochemical detection. We developed a new, simple RT-PCR based assay for detection of EGFRvIII in formalin fixed, paraffin embedded (FFPE) tissues.
Design: From January 2007 to August 2008, 104 cases of newly diagnosed gliomas were retreived from the pathology information system database at our institution. Archival FFPE tissue blocks from 39 patients were selected based on tissue availability and adequate proportion of tumor for RNA extraction on histology review. RNA was exctrated using RecoverAll Total Nucleic Acid Isolation Kit (Ambion, Austin, TX; Applied Biosystems, Foster City, CA). RNA quality and integrity was assesed by RT-PCR for B-actin transcripts. New primers were designed for RT-PCR using two-step RT-PCR method (Qiagen, Valencia, CA). We also used published primers for comparison. PCR products were run on 2.5% agarose gel for detection. All the positive samples were confirmed by bidirectional direct sequencing (BigDye terminator v3.1, Applied Biosystems, Foster City, CA).
Results: There were 19 glioblastomas, 3 anaplastic astrocytomas, 4 low-grade astrocytomas, 9 oligodendrogliomas, and 4 mixed oligoastrocytomas. Our RT-PCR assay detected the EGFRvIII mutation in 7 of 19 (37%) glioblastomas. In contrast, only 3 of these 7 were detected by using published primers. All the 7 positive cases were confirmed by direct sequencing. All the other cases (astrocytomas, oligoastrocytomas, oligodendrogliomas) were negative with either RT-PCR primers.
Conclusions: Our assay provides a simple, semi-quantitative, accurate detection of EGFRvIII mutation from clinical FFPE glioma samples. The increased sensitivity of our assay in the detection of this mutation may be explained by differences in the RNA extraction method and the PCR conditions. This assay may aid in stratifying patients with glioblastomas to predict response to EGFR kinase inhibitor therapy and further help in individualized targeted therapy strategies.
Monday, March 9, 2009 9:30 AM
Poster Session I Stowell-Orbison/Autopsy Award # 222, Monday Morning