PATH-06. A NEW DIAGNOSTIC ASSAY FOR THE DETECTION OF EGFR AMPLIFICATION IN GLIOBLASTOMA TISSUE SPECIMENS

Abstract

Glioblastoma (GBM) is the most common malignant brain tumor among adults with poor prognosis. Epidermal Growth Factor Receptor (EGFR) gene is found to be amplified in approximately 50% of GBM tumors. Drugs that target amplified EGFR are being evaluated in the clinical trials for the treatment of GBM patients. A new Vysis EGFR FISH (Fluorescence in situ Hybridization) assay was developed to detect amplification of the EGFR gene in glioblastoma tissue samples. The assay is comprised of two DNA probes labeled with spectrally distinct fluorophores: orange EGFR probe that hybridizes to 7p11.2-7p12 region, and green CEP 7 probe that hybridizes to a centromere of chromosome 7. Analytical sensitivity and specificity of the probes was tested on 100 metaphase spreads from 5 donors and was shown to be 100%. The assay conditions were optimized in guardbanding studies using universal reagents kits with both manual and automated VP 2000 processing methods. In feasibility study the assay demonstrated 100% (142/142) success rate for manual and 99.3% (141/142) for automated VP 2000 slide processing methods. Analytical reproducibility showed no significant differences between reviewers, slides and laboratories. The positive percent agreement for EGFR amplified specimens was 100% (480/480); negative percent agreement for non-amplified specimens was 99.8% (479/480), and overall percent agreement was 99.9% (959/960). Vysis EGFR CDx FISH Kit demonstrated high photostability, in-use stability, and real time stability for at least 19 months. Stability of FFPE tissue slides was demonstrated that allows using tissue blocks and tissue sections archived for up to 32 months without loss of performance. Fully characterized Vysis EGFR FISH assay proved to be an effective tool in detecting EGFR gene amplification in GBM FFPE tumor tissue with manual and automated assay formats. Vysis EGFR CDx FISH Kit* is being validated in the ongoing clinical trials. *In Development and not Commercially Available