MDM2 RNA In Situ Hybridization for the Diagnosis of Atypical Lipomatous Tumor A Study Evaluating DNA, RNA, and Protein Expression Anupriya S. Kulkarni, PhD,* John B. Wojcik, MD, PhD,† Abhijit Chougule, MD,* Kshitij Arora, MD,* Yashaswini Chittampalli,* Pawel Kurzawa, MD, PhD,‡ John T. Mullen, MD,§ Ivan Chebib, MD,* G. Petur Nielsen, MD,* Miguel N. Rivera, MD,* David T. Ting, MD,8 and Vikram Deshpande, MD* Abstract: The distinction of atypical lipomatous tumor/well- differentiated liposarcoma (ALT/WDL) from its benign counterpart, lipoma, may represent a challenge. MDM2 DNA amplification is used as the gold standard as MDM2 immunohistochemistry lacks specificity and sensitivity. Herein, we investigate the diagnostic utility of MDM2 RNA in situ hybridization (RNA-ISH) and compare the test with MDM2 immunohistochemistry and MDM2 DNA fluo- rescence in situ hybridization (FISH) in benign and malignant lip- omatous neoplasms. We evaluated 109 neoplasms including 27 lipomas, 25 spindle cell lipomas, 32 ALTs/WDLs, and 25 dediffer- entiated liposarcomas (DDL). The validation cohort included 14 lipoma-like neoplasms that lacked unequivocal features of ALT/ WDL and in which MDM2 immunohistochemistry was either equivocal, negative or falsely positive. Immunohistochemistry, au- tomated RNA-ISH and DNA-FISH for MDM2 were performed. Tumors with diffuse nuclear staining or > 50 dots per cell on RNA- ISH were considered positive. All lipomas and lipoma variants were negative for RNA-ISH while all ALTs/WDLs and DDLs were positive. Eighty percent (24/30) and 92% (22/24) of ALTs/WDLs and DDLs were positive for MDM2 immunohistochemistry. Lipomas and its variants were negative for MDM2 amplification; 92% and 100% of ALTs/WDLs and DDLs showed MDM2 DNA amplifi- cation. The mean percentage of ALT/WDL tumor cells showing MDM2 RNA-ISH positivity was 73% compared with 24% on MDM2 immunohistochemistry. RNA-ISH correctly classified all 10 ALTs/WDLs and all 4 lipomas in the validation cohort. The per- formance of MDM2 RNA-ISH and MDM2 DNA-FISH are equivalent. MDM2 RNA-ISH can be of diagnostic value in histo- logically challenging lipomatous neoplasms. The automated MDM2 RNA-ISH assay should allow for more widespread use of MDM2 testing and for a more sensitive and specific diagnosis of ALT/WDL. Key Words: RNA in situ hybridization, MDM2, atypical lip- omatous tumor, well-differentiated liposarcoma, dedifferentiated liposarcoma (Am J Surg Pathol 2019;43:446–454) L iposarcoma, a common soft tissue malignancy, ac- counts for 20% of all sarcomas. 1,2 On the basis of clinical, pathologic, and molecular characteristics the World Health Organization recognizes four subtypes of malignant lipomatous neoplasms: (1) atypical lipomatous tumor (ALT) and well-differentiated liposarcoma (WDL), (2) dedifferentiated liposarcoma (DDL), (3) myxoid lip- osarcoma, and (4) pleomorphic liposarcoma. 3 These sub- types are distinct entities in terms of clinical outcome, tumor location, and expression of molecular markers. 1 The distinction of ALT/WDL from its benign counterpart, lipoma, is generally accomplished on routine histo- pathology. The presence of large atypical hyperchromatic nuclei and lipoblasts, typically embedded in dense col- lagenous tissue, is a hallmark of ALT/WDL. However, in some cases the morphologic distinction between lipoma and ALT/WDL is challenging, often because of poor representation of these atypical cells. This is especially true in the case of biopsies where only limited tissue is available. 2,4 Some lipomatous tumors such as hibernomas and spindle cell lipomas may also mimic ALT/WDL. 5 MDM2 amplification, a characteristic feature of ALT/WDL and DDL, is absent in benign adipocytic tumors. 6–8 Fluorescence in situ hybridization (FISH) for MDM2 is considered the gold standard for distinguishing lipoma from ALT/WDL because of its high sensitivity and specificity. However, DNA-FISH has a number of short- comings which have limited widespread clinical im- plementation of this assay including expense and slow turnaround time. Accordingly, immunohistochemistry for MDM2 protein is more widely utilized, although it has a much lower sensitivity and specificity. 9,10 Other antibodies reported to assist in distinguishing ALT/WDL from lip- oma include CDK4 and p16 and a combination of these From the Departments of *Pathology; 8Medicine, Division of Oncology; §Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA; †Department of Pathology and Laboratory Medicine Perelman, School of Medicine at the University of Pennsylva- nia, Philadelphia, PA; and ‡Department of Clinical Pathology, Poznan University of Medical Sciences, Poznan, Poland. Conflicts of Interest and Source of Funding: M.N.R., D.T.T. and V.D. receive research support from Advanced Cell Diagnostics. The re- maining authors have disclosed that they have no significant rela- tionships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Vikram Deshpande, MD, Department of Pathology, 55 Fruit Street, Warren 2, Boston, MA 02478 (e-mail: vikramdirdeshpande@gmail. com). Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. ORIGINAL ARTICLE 446 | www.ajsp.com Am J Surg Pathol  Volume 43, Number 4, April 2019 Copyright r 2018 Wolters Kluwer Health, Inc. All rights reserved.