Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets

• DOI: 10.18632/oncotarget.20098
• Publication Date: 2017-08-09T20:59:57Z
• Authors: Michaela C. Baldauf; Martin F. Orth; Marlene Dallmayer; Aruna Marchetto; Julia S. Gerke; Rebeca Alba Rubio; Merve M. Kiran; Julian Musa; Maximilian M. L. ...; Shunya Ohmura; Jing Li; Nusret Akpolat; Ayse N. Akatli; Özlem Özen; Uta Dirksen; Wolfgang Hartmann; Enrique de Alava; Daniel Baumhoer; Giuseppina Sannino; Thomas Kirchner; Thomas G. P. Grün...
• Keywords: Ewing-like sarcoma; 03 medical and health sciences; 0302 clinical medicine; immunohistochemistry; Medizin; BCL11B; GLG1; Immunohistochemistry; Ewing sarcoma; Research Paper; 3. Good health

AbstractEwing sarcoma is an undifferentiated bone-associated cancer. Although molecular detection of pathognomonicEWSR1-ETSfusions such asEWSR1-FLI1enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based solely on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. This study aimed to identify novel diagnostic immunohistochemical markers for Ewing sarcoma.We analyzed 768 expression microarrays representing 21 tumor entities including Ewing-like sarcomas to nominate candidate biomarkers. These candidates were validated by immunohistochemistry (IHC) in a tissue microarray (TMA) comprising 174 samples. Microarray, chromatin immunoprecipitation and sequencing (ChIP-Seq) data, and reporter assays were employed to analyze their EWSR1-FLI1-dependency.Our comparative expression analyses revealed thatATP1A1, BCL11B, andGLG1constitute specific markers for Ewing sarcoma. Analysis of ChIP-Seq and microarray datasets showed that their expression is EWSR1-FLI1-dependent. This outcome corresponded to EWSR1-FLI1-binding to proximal super-enhancers, which showed high activity in reporter assays. Consistently, high ATP1A1, BCL11B, and GLG1 expressions were detected by IHC. Automated cut-off-finding and combination-testing in the TMA 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 high expression of BCL11B and GLG1.Collectively, we provide evidence thatATP1A1, BCL11B, andGLG1are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple and cost-efficient way to diagnose Ewing sarcoma by IHC. We anticipate that these markers will significantly reduce the number of misdiagnosed patients, and thus improve patient care.