Evaluation of epidermal growth factor receptor (EGFR) by chromogenic in situ hybridization (CISH™) and immunohistochemistry (IHC) in archival gliomas using bright-field microscopy

Overexpression of EGFR secondary to EGFR gene amplification is a common feature in primary malignant gliomas. To correctly assess EGFR protein and gene level as possible prognostic and predictive markers in gliomas, straightforward assays, which can be used routinely in the pathology laboratory to evaluate EGFR status, becomes critical. EGFR gene amplification and chromosome 7 aneuploidy was detected in 34 formalin-fixed, paraffin-embedded benign and malignant gliomas by chromogenic in situ hybridization (CISH(TM)) using digoxigenin-labeled EGER and biotin-labeled chromosome 7 centromeric probes. The results were evaluated by bright-field microscopy under a 40x objective lens. EGFR protein level was detected by immunohistochemistry (IHC) using monoclonal antibody 31G7. Five cases, 3 astrocytoma grade III (33%) and 2 glioblastoma multiforme (GBM) (33%), had EGFR amplification displayed as diaminobenzidine-stained multiple dots suggesting the pattern of double-minute chromosomes. Chromosome 7 polysomy was found in 68% gliomas, 100% GBM, 67% astrocytoma grade III, 42% astrocytoma grade II, 50% astrocytoma grade 1, 100% ependymoma, and the I case of mixed glioma III. High expression of EGFR protein was present in 62% gliomas and displayed membrane and cytoplasmic staining. All tumors with EGFR gene amplification showed EGFR high expression. High expression of EGER without gene amplification was observed in all grades of gliomas. Simultaneous detection of EGFR gene copies or chromosome 7 centromere signals along with tissue morphology allows us to compare CISH(TM) results easily with IHC results. Our results show that CISH(TM) is an objective, practical, and accurate assay to screen for EGFR gene status in gliomas.