Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets
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Michaela C. Baldauf1,*, Martin F. Orth1,*, Marlene Dallmayer1,*, Aruna Marchetto1, Julia S. Gerke1, Rebeca Alba Rubio1, Merve M. Kiran2, Julian Musa1, Maximilian M. L. Knott1, Shunya Ohmura1, Jing Li1, Nusret Akpolat3, Ayse N. Akatli3, Özlem Özen4, Uta Dirksen5, Wolfgang Hartmann6, Enrique de Alava7, Daniel Baumhoer8, Giuseppina Sannino1, Thomas Kirchner9,10,11 and Thomas G. P. Grünewald1,9,10,11
1 Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany
2 Department of Pathology, Medical Faculty, Ankara Yildirim Beyazit University, Ankara, Turkey
3 Department of Pathology, Turgut Ozal Medical Center, Inonu University, Malatya, Turkey
4 Department of Pathology, Başkent University Hospital, Ankara, Turkey
5 Department of Pediatric Hematology and Oncology, University Hospital Essen, Essen, Germany
6 Gerhard-Domagk-Institute for Pathology, University Hospital Münster, Westfalian Wilhelms University, Münster, Germany
7 Institute of Biomedicine of Seville (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, CIBERONC, Seville, Spain
8 Bone Tumour Reference Center, Institute of Pathology, University Hospital Basel, University of Basel, Basel, Switzerland
9 Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany
10 German Cancer Consortium (DKTK), Heidelberg, Germany
11 German Cancer Research Center (DKFZ), Heidelberg, Germany
* These authors have contributed equally to this work
Thomas G. P. Grünewald, email:
Keywords: Ewing sarcoma, Ewing-like sarcoma, immunohistochemistry, BCL11B, GLG1
Received: July 18, 2017 Accepted: July 23, 2017 Published: August 04, 2017
Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics.
To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity.
Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care.
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