MDM2 Determination by Brightfield Dual Color In Situ Hybridization: Interobserver Reproducibility and Correlation with FISH
Gloria Zhang, Erinn Downs-Kelly, Christopher Lanigan, Raymond Tubbs. Cleveland Clinic, Cleveland, OH
Background: MDM2 gene amplification is present in approximately 20% of sarcomas. The identification of MDM2 amplification can serve as an ancillary diagnostic tool especially in the setting of atypical lipomatous tumors /well-differentiated liposarcoma as they characteristically harbor alterations in the 12q13-15 amplicons resulting in amplification of MDM2; conversely, lipomas and other histologic mimics lack amplification. We have previously described a brightfield assay (AIMM 2011; 19:54-61) to visualize both MDM2 and CHR12 within the same tumor nuclei. Using a third generation repeat-free probe set and dual-color, in situ hybridization (Dual ISH) brightfield detection assay (VentanaMedical Systems, Inc. Tucson AZ) we assessed interobserver interpretative reproducibility of Dual ISH and correlated Dual ISH with the MDM2 genotype as determined by fluorescence in situ hybridization (FISH; Abbott Molecular Vysis).
Design: Lipomatous lesions seen in a soft tissue consultative practice were assessed for MDM2 amplification by both FISH and Dual ISH on consecutive 4 micron whole tissue sections of formalin-fixed and paraffin-embedded tissue. Fluorescent and brightfield microscopy were used respectively with the average number of MDM2 and CHR12 signals enumerated in the nuclei by quantification of at least 20 nuclei/specimen with a MDM2/CHR12 ratio calculated for each case. A ratio of <2.0 was considered non-amplified whereas a ratio ≥ 2.0 was considered amplified, according to previously published criteria. Cases with endogenous signal present in endothelial cells or fibroblasts were considered scorable and were scored independently by two reviewers; correlation between observers and FISH was assessed.
Results: Of the 56 cases assayed, FISH and Dual ISH had complete concordance (100%) with 16 cases identified as MDM2 amplified and 40 cases identified as MDM2 non-amplified. Agreement between observers (EDK and RRT) was similarly excellent with all scored cases being concordant.
Conclusions: Brightfield Dual ISH assessment of MDM2 gene status has excellent correlation with FISH and is reproducible among reviewers. This assay offers the advantage of brightfield microscopy which allows for the assessment of tissue morphology which can be extremely helpful in cases where atypical/neoplastic nuclei are limited.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 272, Wednesday Morning