Stratification of patients based on EGFR gene status in a stratified phase II study of cetuximab for the treatment of recurrent glioblastoma multiforme

Background: The EGFR-gene is frequently amplified in high-grade gliomas. We are investigating the activity of the EGFR-targeted antibody Cetuximab for treatment of patients with recurrent glioblastoma multiforme following surgery, radiotherapy and chemotherapy. Patients with recurrent GBM are treated in two parallel treatment arms according to the amplification status of the EGFR-gene. Patients are enrolled according to a Simon two-stage phase II study design, applied to both study arms separately.
Material and methods: The EGFR-gene status is analysed on formalin fixed/paraffin embedded archival glioma tissue (Vysis LSI EGFR/CEP7 probe set). Slides are immersed three times in xylene and twice in 100% ethanol. After air-drying, slides are put in 0,2N HCl for 20'. After washing in distilled water and 2xSCC, slides are immersed in pretreatment solution (80C) for 30'. Slides are washed in distilled water and 2xSSC before digestion in protease solution at 37C and washing in 2xSSC. Denaturing: 5' in a mix of 49ml formamide, 7 ml 20xSSC, 14 ml distilled water (72C, pH 7,5). After dehydrating in graded ethanol, slides are dried. The probe mix is applied and probes are hybridized to the EGFR-gene overnight (37C). Slides are put in posthybridization buffer (2xSSC/0,3% NP-40; pH 7,25) at 72C for 2' DAPI counterstain is applied after drying in the dark for 30'. Slides are evaluated under a fluorescence
microscope. Ratios of EGFR-gene copy number to a reference signal (centromere chromosome 7) are calculated.
Results: At present, glioma tissues from 41 patients have been screened. Recruitment of non-amplified patients has been completed (efficacy analysis ongoing), while recruitment of amplified patients is ongoing. Fourteen patients (34,1%) were found to have EGFR-amplification (ratios = 3,7-12,77) and 27 (65,9%) patients had no EGFRamplification (ratios: 0,87-1,62). In four patients two metachronous tissue samples were examined. Results were concordant in three patients while in one amplification found at diagnosis was no longer present at recurrence.
Conclusions: EGFR FISH is a reliable method to be used on archival glioma tissue to
stratify patients in prospective studies with EGFR inhibitors. Such stratification should
be helpful to interpret activity of the study drug within the context of the predefined
genetic background.