Fully Automated Dual ISH (Dual-Color Dual-Hapten Silver In Situ Hybridization) for EGFR Amplification in Glioblastomas
Zhen Wang, Bryce Portier, Christopher Lanigan, Raymond R Tubbs. Cleveland Clinic, Cleveland, OH
Background: EGFR amplification occurs in 40-60% of primary glioblastomas and is typically detected by fluorescence in situ hybridization (FISH). In this study, detection of EGFR amplification by automated dual-color dual-hapten silver ISH (DDISH) was compared to FISH in a series of 73 glioblastomas.
Design: FISH testing was performed on paraffin embedded formalin fixed tissue using a commercially available EGFR ASR probe paired with a CEP7 centromeric control probe (Abbott Molecular) In addition to FISH each case was analyzed independently by DDISH. DDISH staining was performed using the Dual Color Open Probe software on a Ventana Benchmark XT. DNP labeled/repeat depleted EGFR and Digoxigenin labeled Chromosome 7 (CHR7) probes were utilized. Pretreatment conditions were: Extended II deparaffinization at 72°C, 3 cycles of cell conditioning with CC2 for 12 minutes at 80°C, and protease digestion with ISH protease 3 for 32 minutes. Denaturation was at 80°C for 12 minutes; hybridization at 44°C for 6 hours; followed by stringency washes (3 at 72°C, 8 minutes). EGFR DNP probe was detected with ultraView silver ISH (SISH) DNP Detection Kit: silver anti-hapten antibody (20 minutes), and SISH detection (8 minutes). CHR7 DIG probe was detected with ultraView Red ISH DIG Detection Kit: anti-hapten antibody incubated (20 minutes) and Red detection (8 minutes). The slides were counterstained with hematoxylin II for 8 minutes, post counterstained with bluing reagent for 4 minutes, and mounted as permanent slides for bright field microscopy. Metallic black silver (EGFR) and reference CHR7 red signals were qualitatively and semi-quantitatively enumerated for tumor nuclei. Small and large clusters of silver signals were recorded as 6 or 12 signals respectively. EGFR amplification was defined as EGFR/CHR7 ratio >2.0.
Results: Evaluable signals with amplified (n=29) and non-amplified (n=40) DDISH were obtained in 69 cases (95%). Concordance between FISH and DDISH was observed for all cases. FISH-defined EGFR counts (mean 17.4 vs. 2.7) and EGFR/CEP7 ratios (mean 6.0 vs. 1.1) for DDISH amplified and DDISH non-amplified cases were significantly different (p<0.0001).
Conclusions: In this representative series of glioblastomas, utilization of DDISH correctly identified all EGFR amplified cases as previously assessed by FISH. These data suggest that DDISH can be utilized as an ancillary tool for detecting EGFR amplificantion and therefore may be useful in molecular characterization of gliomas.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 255, Tuesday Afternoon