Determination of EGFR, Her2/neu, PTEN, and p53 protein expression and gene status by immunohistochemistry and fluorescence in situ hybridization in high-grade glioma

Background: The EGFR, Her2/neu, PTEN, and p53 genes play an important role in the pathogenesis and biology of high-grade glioma (HGG, WHO grade III and IV). All of them are either involved through
amplification, loss of heterozygosity, and/or mutation. Immunohistochemistry (IHC) and FISH tests are commercially available to determine the expression and copy number status of all four genes. This study aimed to determine the usefulness of IHC and FISH and to establish the correlation between both tests for these genes in an HGG cohort. This knowledge should be of help in stratifying glioma patients in clinical trials with targeted agents.
Materials and methods: Tumor tissues were collected from 72 patients from six Belgian hospitals. In addition to conventional histopathology, tumors were characterized by IHC for expression of the EGFR (DAKO, K1492), Her2/neu (DAKO, K5204), PTEN (Cascade Biosciences, ABM-2052), and p53 (Novocastra, NCL-p53 - DO7); and by FISH for the copy number status for EGFR (Vysis, 32 - 191053), Her2/neu (Vysis, 30 - 161060), PTEN (Vysis, 32 - 231010), and p53 (Vysis, 32 - 190008).
Results: The patient population (n 5 72) consisted of 49 men and 23 women with a median age of 55 years at diagnosis. According to WHO classification, there were 23 anaplastic astrocytomas (AAs) and 49 glioblastomas multiforme (GBMs). None of the gliomas in this series expressed Her2/neu. FISH has revealed no altered Her2/neu gene copy number in 16 of 16 gliomas examined so far. FISH identified an amplification of the EGFR gene in 4 of 23 AAs (17%) and in 16 of 49 GBMs (33%). Interpretation of FISH results was straightforward. A positive EGFR IHC staining was observed in 18 of 23 AAs (78%) and 43 of 49 GBMs (88%) when a cutoff of 10% positive cells with membrane staining taken into account. EGFR
gene amplification did not correlate with the level of expression assessed by IHC staining. Analysis of the PTEN (FISH and IHC) and P53 (FISH and IHC) genes is ongoing.
Conclusions: The Her2/neu proto-oncogene is not expressed in AA and GBM, and we found no alternation of the gene copy number so far. Therefore, we assume no important role for this gene in the biology of HGG. We confirmed that EGFR is expressed in most AAs and GBMs and that the EGFR gene is frequently amplified in these tumors. EGFR IHC results did not correlate with EGFR gene amplification status as determined by FISH. Stratification of glioma patients in clinical trials according to EGFR FISH results seems feasible and preferable as compared to IHC.