Are EWSR1-NFATc2-positive sarcomas really Ewing sarcomas?
2018; Elsevier BV; Volume: 31; Issue: 6 Linguagem: Inglês
10.1038/s41379-018-0009-7
ISSN1530-0285
AutoresMichaela C. Baldauf, Julia S. Gerke, Martin F. Orth, Marlene Dallmayer, Daniel Baumhoer, Enrique de Álava, Wolfgang Hartmann, Thomas Kirchner, Thomas G. P. Grünewald,
Tópico(s)Cardiac tumors and thrombi
ResumoRecently, Charville et al. [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar] reported that EWSR1-rearranged fusion proteins mediate the expression of the paired-box transcription factor PAX7 in Ewing sarcoma. Based on an analysis of a published gene expression microarray dataset (accession code GSE60740), they state having identified PAX7 to be significantly overexpressed in Ewing sarcoma in comparison to CIC-DUX4-positive round cell sarcomas [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. In that microarray analysis they compared CIC-DUX4-positive sarcomas with EWSR1-NFATc2-positive sarcomas, assuming that EWSR1-NFATc2-positive sarcomas belong to the family of Ewing sarcomas [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar], which are typically characterized by EWSR1-ETS fusion oncogenes [2Kovar H Amatruda J Brunet E et al.The second European interdisciplinary Ewing sarcoma research summit—a joint effort to deconstructing the multiple layers of a complex disease.Oncotarget. 2016; 7: 8613-862410.18632/oncotarget.6937Crossref PubMed Scopus (36) Google Scholar]. Accordingly, Charville et al. [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar] summarized in a schematic EWSR1-FLI1-, EWSR1-ERG-, and EWSR1-NFATc2-positive sarcomas as "Ewing sarcoma," referring to Szuhai et al. (2009) [3Szuhai K Ijszenga M de Jong D et al.The NFATc2 gene is involved in a novel cloned translocation in a Ewing sarcoma variant that couples its function in immunology to oncology.Clin Cancer Res. 2009; 15: 2259-226810.1158/1078-0432.CCR-08-2184Crossref PubMed Scopus (156) Google Scholar], and did not take into account more recent reports in the literature that EWSR1-NFATc2-positive sarcomas may constitute an own entity [2Kovar H Amatruda J Brunet E et al.The second European interdisciplinary Ewing sarcoma research summit—a joint effort to deconstructing the multiple layers of a complex disease.Oncotarget. 2016; 7: 8613-862410.18632/oncotarget.6937Crossref PubMed Scopus (36) Google Scholar]. Comparison of the dataset (GSE60740) used by Charville et al. to a published transcriptome reference dataset of genetically defined EWSR1-ETS-positive Ewing sarcomas (GSE34620) [4Postel-Vinay S Véron AS Tirode F et al.Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma.Nat Genet. 2012; 44: 323-32710.1038/ng.1085Crossref PubMed Scopus (139) Google Scholar] and 13 other malignancies that may constitute morphological mimics [5Baldauf MC Orth MF Dallmayer M et al.Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets.Oncotarget. 2018; 5: 1587-1601Crossref Scopus (46) Google Scholar] shows that EWSR1-NFATc2-positive sarcomas do not cluster with any other analyzed tumor entity including EWSR1-ETS-positive Ewing sarcoma (Fig. 1a). Furthermore, Charville et al. noted that (in addition to PAX7) the genes FOXG1, NR5A2, SOX5, VDR, and TFAP2 were highly overexpressed in EWSR1-NFATc2-positive sarcomas as compared to CIC-DUX4-positive sarcomas [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Interestingly, all these genes are similarly lowly expressed in EWSR1-ETS-positive Ewing sarcomas and CIC-DUX4-positive sarcomas (Fig. 1b). Collectively, these analyses strongly suggest that EWSR1-NFATc2-positive sarcomas constitute a tumor entity distinct from Ewing sarcoma [5Baldauf MC Orth MF Dallmayer M et al.Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets.Oncotarget. 2018; 5: 1587-1601Crossref Scopus (46) Google Scholar]. Hence, the correctness of extrapolation from EWSR1-NFATc2-positive sarcomas to Ewing sarcoma remains to be substantiated. Based on their disputable extrapolation from EWSR1-NFATc2-positive sarcomas to Ewing sarcomas, Charville et al. [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar] performed immunohistochemical analyses of tissue samples, which were "diagnosed as Ewing sarcoma using a combination of morphologic, immunohistochemical, and molecular features." For the majority of cases (102/103), which were all "PAX7-positive" by immunohistochemistry, no details or references were provided on how the diagnosis of Ewing sarcoma was molecularly confirmed. Only for one sample, which was classified as "PAX7-negative," details were provided, but the diagnosis of Ewing sarcoma could not be molecularly confirmed [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. The authors conclude from this sample set that "immunohistochemical detection of anti-PAX7 immunohistochemistry is a sensitive marker for Ewing sarcoma" [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Yet, it remains unclear whether the sensitivity of PAX7 is superior to that of the established Ewing sarcoma marker CD99 [6Shibuya R Matsuyama A Nakamoto M et al.The combination of CD99 and NKX2.2, a transcriptional target of EWSR1-FLI1, is highly specific for the diagnosis of Ewing sarcoma.Virchows Arch. 2014; 465: 599-60510.1007/s00428-014-1627-1Crossref PubMed Scopus (67) Google Scholar]. In their transcriptome analysis of EWSR1-NFATc2-positive sarcomas, Charville et al. noted that CD99 was not significantly overexpressed compared to CIC-DUX4-positive sarcomas [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar], supporting the concept that EWSR1-NFATc2-positive sarcomas are distinct from Ewing sarcoma, which typically shows high and uniform CD99 expression [6Shibuya R Matsuyama A Nakamoto M et al.The combination of CD99 and NKX2.2, a transcriptional target of EWSR1-FLI1, is highly specific for the diagnosis of Ewing sarcoma.Virchows Arch. 2014; 465: 599-60510.1007/s00428-014-1627-1Crossref PubMed Scopus (67) Google Scholar]. The proposed utility of PAX7 as a marker for Ewing sarcoma might be diminished given its limited specificity: In a previous report Charville et al. showed very high expression of PAX7 in morphological mimics such as embryonal, alveolar, and pleomorphic rhabdomyosarcomas, as well as synovial sarcomas [7Charville GW Varma S Forgó E et al.PAX7 expression in rhabdomyosarcoma, related soft tissue tumors, and small round blue cell neoplasms.Am J Surg Pathol. 2016; 40: 1305-131510.1097/PAS.0000000000000717Crossref PubMed Scopus (30) Google Scholar], and in the current report they provide first evidence for its high expression in EWSR1-NFATc2-positive sarcomas [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Though PAX7 has been shown to be highly expressed in morphological mimics not positive for EWSR1-regarrangements such as rhabdomyosarcoma and synovial sarcoma [7Charville GW Varma S Forgó E et al.PAX7 expression in rhabdomyosarcoma, related soft tissue tumors, and small round blue cell neoplasms.Am J Surg Pathol. 2016; 40: 1305-131510.1097/PAS.0000000000000717Crossref PubMed Scopus (30) Google Scholar], Charville et al. explain the high expression of PAX7 in "Ewing sarcoma" by EWSR1-FLI1 binding to a GGAA-microsatellite sequence located 20 kb telomeric to the PAX7 gene [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar], which shows epigenetic characteristics of an active enhancer element in published ChIP-Seq data [8Riggi N Knoechel B Gillespie SM et al.EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma.Cancer Cell. 2014; 26: 668-68110.1016/j.ccell.2014.10.004Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar]. However, binding of EWSR1-FLI1 to this DNA sequence, EWSR1-FLI1-dependent enhancer activity, and a true interaction of this potential enhancer with the PAX7 promoter were not confirmed experimentally. Yet, Charville et al. deduce from their approach that "these analyses provide evidence that a cis regulatory mechanism of EWSR1-FLI1 binding and enhancer activation leads to expression of PAX7 in Ewing sarcoma" [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. In the corresponding figure, two additional and more proximal EWSR1-FLI1-bound DNA elements are shown (the first mapping to another GGAA microsatellite, the second to a canonical ETS-like binding motif) with EWSR1-FLI1-dependent epigenetic characteristics of active enhancers, which were not further investigated or discussed by the authors [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Given that there are thousands of such EWSR1-FLI1-bound GGAA microsatellites in the vicinity of genes in Ewing sarcoma tumors [8Riggi N Knoechel B Gillespie SM et al.EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma.Cancer Cell. 2014; 26: 668-68110.1016/j.ccell.2014.10.004Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar 9Tomazou EM Sheffield NC Schmidl C et al.Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1.Cell Rep. 2015; 10: 1082-109510.1016/j.celrep.2015.01.042Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar], it appears questionable to conclude that any EWSR1-FLI1-bound enhancer actually represents the cis regulatory element controlling the expression of a certain nearby gene. Previous reports showed that such EWSR1-FLI1-bound GGAA microsatellites can loop to and accordingly regulate genes located in great distances (more than 300 kb) [8Riggi N Knoechel B Gillespie SM et al.EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma.Cancer Cell. 2014; 26: 668-68110.1016/j.ccell.2014.10.004Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar]. Thus, the potential enhancer reported by Charville et al. could control the expression of any gene in cis or trans. Taking into account that GGAA microsatellites underlie substantial germline variability and that the enhancer activity of EWSR1-ETS-bound GGAA microsatellites strongly depends on the number of consecutive GGAA-repeats [10Grünewald TGP Bernard V Gilardi-Hebenstreit P et al.Chimeric EWSR1-FLI1 regulates the Ewing sarcoma susceptibility gene EGR2 via a GGAA microsatellite.Nat Genet. 2015; 47: 1073-107810.1038/ng.3363Crossref PubMed Scopus (124) Google Scholar, 11Beck R Monument MJ Watkins WS et al.EWS/FLI-responsive GGAA microsatellites exhibit polymorphic differences between European and African populations.Cancer Genet. 2012; 205: 304-31210.1016/j.cancergen.2012.04.004Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 12Monument MJ Johnson KM Mcllvaine E et al.Clinical and biochemical function of polymorphic NR0B1 GGAA-microsatellites in Ewing sarcoma: a report from the Children's Oncology Group.PLoS ONE. 2014; 9: e10437810.1371/journal.pone.0104378Crossref PubMed Scopus (31) Google Scholar], it seems improbable that a highly polymorphic enhancer-like DNA element should lead to a uniformly high expression of PAX7 in Ewing sarcoma as reported by Charville et al. [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Nevertheless, the authors conclude that EWSR1 fusion proteins are required for PAX7 expression in Ewing sarcoma by this mechanism. Given that ChIP-Seq analyses of Charville et al. are restricted to EWSR1-FLI1 [1Charville GW Wang WL Ingram DR et al.EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.Mod Pathol. 2017; 30: 1312-132010.1038/modpathol.2017.49Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar], that there is as yet no convincing evidence that EWSR1-NFATc2 can bind to GGAA microsatellites as well, and that immunohistochemical analysis of PAX7 on EWSR1-NFATc2-positive sarcomas has not been performed, this conclusion is critical. We deduce from the report of Charville et al. that particular care must be taken when analyzing published "omics"-data before conclusions may be drawn. The authors declare that they have no conflict of interest. PAX7 expression in sarcomas bearing the EWSR1-NFATC2 translocationModern PathologyVol. 32Issue 1PreviewWe thank Dr. Grunewald and colleagues for reading our manuscript [1], and we are happy to respond to their comments. In their letter, Grunewald et al. [2] raise the question of whether EWSR1-NFATC2-positive sarcomas really are Ewing sarcomas. They provide information from their interesting publication that appeared in Oncotarget to support the idea that EWSR1-NFATC2-positive sarcomas are transcriptionally distinct from tumors with EWSR1-FLI1 translocation. Unfortunately, their manuscript was published after the publication of our paper, and therefore we could not reference their data in our report. Full-Text PDF Open Access
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