Comparison of automated silver enhanced in situ hybridization and fluorescence in situ hybridization for evaluation of epidermal growth factor receptor status in human glioblastomas

Abstract

The epidermal growth factor receptor (EGFR) is amplified in approximately 40% of glioblastomas making it a compelling molecular target for therapy. Before starting a therapy targeting the EGFR pathway, accurate determining of EGFR status is a prerequisite. We evaluated the reliability of the novel automated silver enhanced in situ hybridization for the detection of EGFR gene amplification in human glioblastomas. EGFR-amplification status was assessed in 93 cases of glioblastoma by silver enhanced in situ hybridization and compared with results of fluorescence in situ hybridization and immunohistochemistry. In a second cohort, silver enhanced in situ hybridization status was correlated with EGFR gene expression data. The EGFR gene was amplified in 25/90 tumours (28%) by silver enhanced in situ hybridization, and in 28/93 tumours (30%) by fluorescence in situ hybridization. The concordance rate for silver enhanced in situ hybridization and fluorescence in situ hybridization was 98%. Two glioblastomas were scored as being amplified by fluorescence in situ hybridization but not by silver enhanced in situ hybridization. Polymerase chain reaction-based EGFR-amplification data were highly correlated with EGFR silver enhanced in situ hybridization. Altogether, 81 of 91 cases (89%) showed positivity for EGFR expression by immunohistochemistry. Although EGFR protein over expression was associated with gene amplification (r=0.40, P<0.001), there were 29 of 91 cases that showed a high EGFR protein level and no EGFR amplification by fluorescence in situ hybridization. The high concordance rate of silver enhanced in situ hybridization and fluorescence in situ hybridization for the detection of EGFR amplification in paraffin-embedded glioblastomas samples demonstrates that silver enhanced in situ hybridization is a valid and attractive alternative to fluorescence in situ hybridization. Silver enhanced in situ hybridization combines the advantages of bright field microscopy with fully automated analysis in a cost-effective way thereby emphasizing its use for routine application in surgical pathology.