Automated Brightfield Dual Color In Situ Hybridization for Detection of MDM2 Gene Amplification in Sarcomas
W Zhang, A McElhinny, A Nielsen, M Wang, M Miller, S Singh, R Rueger, B Rubin, RR Tubbs, P Roche, P Wu, L Pestic-Dragovich. Ventana Medical Systems, Inc., Tucson, AR; Roche Diagnostics GmbH, Penzberg, Germany; Cleveland Clinic, Cleveland, OH
Background: The human homologue of the mouse double minute 2 (MDM2) oncogene is amplified in about 20% of sarcomas. Measurement of MDM2 amplification can aid in classification, and may provide predictive value for recently formulated therapies targeting MDM2. Here, we have developed and validated an automated brightfield dual color in situ hybridization application for MDM2 gene amplification in sarcomas.
Design: A repeat-depleted MDM2 probe was constructed to target the MDM2 gene region at 12q15. A chromosome 12-specific probe (Chr 12) was generated from pα12H8 plasmid. The ISH assay was developed with dinitrophenyl-labeled MDM2 probe and digoxigenin-labeled Chr 12 probe on Ventana Medical Systems, Inc. automated slide staining platforms. The specificity of the MDM2 and Chr 12 probes was demonstrated on metaphase spreads, and further validated against controls including normal human tonsil and known MDM2-amplified samples. The assay performance was evaluated on a cohort of 68 FFPE specimens using a conventional brightfield microscope.
Results: Simultaneous hybridization and signal detection for MDM2 and Chr 12 demonstrated both DNA targets in the same cells. 66 of 68 cases had interpretable signals for MDM2 and Chr 12. While all four lipomas were nonamplified and eusomic, MDM2 amplification was noted in 81% (21/26) of well-differentiated liposarcomas. MDM2 amplification was observed in 1 of 8 osteosarcomas; 3 demonstrated Chr 12 aneusomy; and the other 4 were non-amplified. MDM2 amplification was present in 1 of 4 chondrosarcomas; the remaining 3 cases had normal MDM2 and Chr 12 copy numbers. 11 of 13 synovial sarcomas displayed no evidence of MDM2 amplification on most cells; the other two had Chr 12 aneusomy. In pleomorphic sarcoma, not otherwise specified (malignant fibrous histiocytoma), MDM2 was amplified in 31% (4/12) interpretable cases, while 92% (11/12) were aneusomy for Chr 12 . One alveolar rhabdomyosarcoma and two embryonal rhabdomyosarcoma cases had low level aneusomy of Chr 12.
Conclusions: The use of ISH MDM2/Chr 12 assay allows the simultaneous analyses of the two DNA targets within the context of tissue morphology. This method combines the advantage of brightfield microscopy with fully automated analysis and has the potential for routine application in surgical pathology.
Monday, March 22, 2010 1:00 PM
Poster Session II # 253, Monday Afternoon