Automated brightfield dual-color in situ hybridization for detection of mouse double minute 2 gene amplification in sarcomas

Wenjun Zhang, Abigail McElhinny, Alma Nielsen, Maria Wang, Melanie Miller, Shalini Singh, Ruediger Rueger, Brian P. Rubin, Zhen Wang, Raymond R. Tubbs, Raymond B Nagle, Pat Roche, Ping Wu, Lidija Pestic-Dragovich

Research output: Contribution to journalArticle

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Abstract

Background: The human homolog of the mouse double minute 2 (MDM2) oncogene is amplified in about 20% of sarcomas. The measurement of the MDM2 amplification can aid in classification and may provide a predictive value for recently formulated therapies targeting MDM2. We have developed and validated an automated bright field dual-color in situ hybridization application to detect MDM2 gene amplification. DESIGN: A repeat-depleted MDM2 probe was constructed to target the MDM2 gene region at 12q15. A chromosome 12-specific probe (CHR12) was generated from a pα12H8 plasmid. The in situ hybridization assay was developed by using a dinitrophenyl-labeled MDM2 probe and a digoxigenin-labeled CHR12 probe on the Ventana Medical Systems' automated slide-staining platforms. The specificity of the MDM2 and CHR12 probes was shown 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 100 formalin-fixed, paraffin-embedded specimens by using a conventional bright field microscope. Results: Simultaneous hybridization and signal detection for MDM2 and CHR12 showed that both DNA targets were present in the same cells. One hundred soft tissue specimens were stained for MDM2 and CHR12. Although 26 of 29 lipomas were nonamplified and eusomic, MDM2 amplification was noted in 78% of atypical lipomatous tumors or well-differentiated liposarcomas. Five of 6 dedifferentiated liposarcoma cases were amplified for MDM2. MDM2 amplification was observed in 1 of 8 osteosarcomas; 3 showed CHR12 aneusomy. MDM2 amplification was present in 1 of 4 chondrosarcomas. Nine of 10 synovial sarcomas displayed no evidence of MDM2 amplification in most tumor cells. In pleomorphic sarcoma, not otherwise specified (pleomorphic malignant fibrous histiocytoma), MDM2 was amplified in 38% of cases, whereas 92% were aneusomic for CHR12. One alveolar rhabdomyosarcoma and 2 embryonal rhabdomyosarcomas displayed low-level aneusomy of CHR12 without net MDM2 amplifications. Conclusions: These results show that the use of the ISH MDM2 and CHR12 assay allows simultaneous analyses of the 2 DNA targets within the context of tissue morphology. This method combines the advantages of a fully automated protocol with bright field microscopy and has the potential for routine application in surgical pathology.

Original languageEnglish (US)
Pages (from-to)54-61
Number of pages8
JournalApplied Immunohistochemistry and Molecular Morphology
Volume19
Issue number1
DOIs
StatePublished - Jan 2011

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Gene Amplification
Sarcoma
In Situ Hybridization
Color
Chromosomes, Human, Pair 12
Liposarcoma
Alveolar Rhabdomyosarcoma
Synovial Sarcoma
Digoxigenin
Malignant Fibrous Histiocytoma
Surgical Pathology
Chondrosarcoma
Lipoma
Palatine Tonsil
DNA

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Medical Laboratory Technology
  • Histology

Cite this

Automated brightfield dual-color in situ hybridization for detection of mouse double minute 2 gene amplification in sarcomas. / Zhang, Wenjun; McElhinny, Abigail; Nielsen, Alma; Wang, Maria; Miller, Melanie; Singh, Shalini; Rueger, Ruediger; Rubin, Brian P.; Wang, Zhen; Tubbs, Raymond R.; Nagle, Raymond B; Roche, Pat; Wu, Ping; Pestic-Dragovich, Lidija.

In: Applied Immunohistochemistry and Molecular Morphology, Vol. 19, No. 1, 01.2011, p. 54-61.

Research output: Contribution to journalArticle

Zhang, W, McElhinny, A, Nielsen, A, Wang, M, Miller, M, Singh, S, Rueger, R, Rubin, BP, Wang, Z, Tubbs, RR, Nagle, RB, Roche, P, Wu, P & Pestic-Dragovich, L 2011, 'Automated brightfield dual-color in situ hybridization for detection of mouse double minute 2 gene amplification in sarcomas', Applied Immunohistochemistry and Molecular Morphology, vol. 19, no. 1, pp. 54-61. https://doi.org/10.1097/PAI.0b013e3181ee8e14
Zhang, Wenjun ; McElhinny, Abigail ; Nielsen, Alma ; Wang, Maria ; Miller, Melanie ; Singh, Shalini ; Rueger, Ruediger ; Rubin, Brian P. ; Wang, Zhen ; Tubbs, Raymond R. ; Nagle, Raymond B ; Roche, Pat ; Wu, Ping ; Pestic-Dragovich, Lidija. / Automated brightfield dual-color in situ hybridization for detection of mouse double minute 2 gene amplification in sarcomas. In: Applied Immunohistochemistry and Molecular Morphology. 2011 ; Vol. 19, No. 1. pp. 54-61.
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abstract = "Background: The human homolog of the mouse double minute 2 (MDM2) oncogene is amplified in about 20{\%} of sarcomas. The measurement of the MDM2 amplification can aid in classification and may provide a predictive value for recently formulated therapies targeting MDM2. We have developed and validated an automated bright field dual-color in situ hybridization application to detect MDM2 gene amplification. DESIGN: A repeat-depleted MDM2 probe was constructed to target the MDM2 gene region at 12q15. A chromosome 12-specific probe (CHR12) was generated from a pα12H8 plasmid. The in situ hybridization assay was developed by using a dinitrophenyl-labeled MDM2 probe and a digoxigenin-labeled CHR12 probe on the Ventana Medical Systems' automated slide-staining platforms. The specificity of the MDM2 and CHR12 probes was shown 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 100 formalin-fixed, paraffin-embedded specimens by using a conventional bright field microscope. Results: Simultaneous hybridization and signal detection for MDM2 and CHR12 showed that both DNA targets were present in the same cells. One hundred soft tissue specimens were stained for MDM2 and CHR12. Although 26 of 29 lipomas were nonamplified and eusomic, MDM2 amplification was noted in 78{\%} of atypical lipomatous tumors or well-differentiated liposarcomas. Five of 6 dedifferentiated liposarcoma cases were amplified for MDM2. MDM2 amplification was observed in 1 of 8 osteosarcomas; 3 showed CHR12 aneusomy. MDM2 amplification was present in 1 of 4 chondrosarcomas. Nine of 10 synovial sarcomas displayed no evidence of MDM2 amplification in most tumor cells. In pleomorphic sarcoma, not otherwise specified (pleomorphic malignant fibrous histiocytoma), MDM2 was amplified in 38{\%} of cases, whereas 92{\%} were aneusomic for CHR12. One alveolar rhabdomyosarcoma and 2 embryonal rhabdomyosarcomas displayed low-level aneusomy of CHR12 without net MDM2 amplifications. Conclusions: These results show that the use of the ISH MDM2 and CHR12 assay allows simultaneous analyses of the 2 DNA targets within the context of tissue morphology. This method combines the advantages of a fully automated protocol with bright field microscopy and has the potential for routine application in surgical pathology.",
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AU - Wang, Maria

AU - Miller, Melanie

AU - Singh, Shalini

AU - Rueger, Ruediger

AU - Rubin, Brian P.

AU - Wang, Zhen

AU - Tubbs, Raymond R.

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AU - Roche, Pat

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N2 - Background: The human homolog of the mouse double minute 2 (MDM2) oncogene is amplified in about 20% of sarcomas. The measurement of the MDM2 amplification can aid in classification and may provide a predictive value for recently formulated therapies targeting MDM2. We have developed and validated an automated bright field dual-color in situ hybridization application to detect MDM2 gene amplification. DESIGN: A repeat-depleted MDM2 probe was constructed to target the MDM2 gene region at 12q15. A chromosome 12-specific probe (CHR12) was generated from a pα12H8 plasmid. The in situ hybridization assay was developed by using a dinitrophenyl-labeled MDM2 probe and a digoxigenin-labeled CHR12 probe on the Ventana Medical Systems' automated slide-staining platforms. The specificity of the MDM2 and CHR12 probes was shown 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 100 formalin-fixed, paraffin-embedded specimens by using a conventional bright field microscope. Results: Simultaneous hybridization and signal detection for MDM2 and CHR12 showed that both DNA targets were present in the same cells. One hundred soft tissue specimens were stained for MDM2 and CHR12. Although 26 of 29 lipomas were nonamplified and eusomic, MDM2 amplification was noted in 78% of atypical lipomatous tumors or well-differentiated liposarcomas. Five of 6 dedifferentiated liposarcoma cases were amplified for MDM2. MDM2 amplification was observed in 1 of 8 osteosarcomas; 3 showed CHR12 aneusomy. MDM2 amplification was present in 1 of 4 chondrosarcomas. Nine of 10 synovial sarcomas displayed no evidence of MDM2 amplification in most tumor cells. In pleomorphic sarcoma, not otherwise specified (pleomorphic malignant fibrous histiocytoma), MDM2 was amplified in 38% of cases, whereas 92% were aneusomic for CHR12. One alveolar rhabdomyosarcoma and 2 embryonal rhabdomyosarcomas displayed low-level aneusomy of CHR12 without net MDM2 amplifications. Conclusions: These results show that the use of the ISH MDM2 and CHR12 assay allows simultaneous analyses of the 2 DNA targets within the context of tissue morphology. This method combines the advantages of a fully automated protocol with bright field microscopy and has the potential for routine application in surgical pathology.

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