EWSR1 Fusions With CREB Family Transcription Factors Define a Novel Myxoid Mesenchymal Tumor With Predilection for Intracranial Location

Abstract

Recurrent gene fusions involving EWSR1 with members of the cAMP response element binding protein (CREB) family (ATF1 and CREB1) have been reported in a diverse group of tumors including angiomatoid fibrous histiocytoma (AFH), soft tissue and gastrointestinal clear cell sarcoma, primary pulmonary myxoid sarcoma, and hyalinizing clear cell carcinoma of salivary gland. We have recently encountered a group of 5 myxoid mesenchymal tumors positive for EWSR1 fusions with one of the CREB family member (ATF1, CREB1, and CREM), with histologic features distinct from any of the previously described pathologic entities. Tumors occurred in children or young adults (12 to 23 y; mean, 18 y), with equal sex distribution. All except 1 were intracranial (intra-axial, 2; meningeal, 2), whereas 1 was perirectal. Histologically, the tumors were well circumscribed, often lobulated, composed of uniform ovoid to round cells, and arranged in cord-like or reticular structures in a myxoid background. All except 1 displayed unique sunburst amianthoid fibers. Immunohistochemically, tumors were positive for epithelial membrane antigen (5/5; 4 focal, 1 diffuse) and desmin (3/5). A novel EWSR1-CREM fusion was identified by RNA sequencing in the perirectal tumor, which was further confirmed by fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR). A second case with similar EWSR1-CREM fusion was identified by RT-PCR and FISH in a meningeal tumor. The remaining cases studied by FISH showed the presence of EWSR1-CREB1 fusion in 2 cases and EWSR1-ATF1 in 1. In conclusion, we report a distinct group of myxoid mesenchymal neoplasms occurring in children or young adults with a predilection for intracranial locations. Although the immunoprofile [epithelial membrane antigen (EMA), desmin] and the fusion type raise the possibility of a myxoid AFH, none of the typical histologic findings of AFH were present, suggesting a novel entity.

Figure 1. A novel EWSR1-CREM gene fusion was identified by transcriptome sequencing in index case 1

(A) Fusion junction reads of paired-end RNAseq identified a candidate t(10;22) translocation resulting in an in-frame fusion between EWSR1 exon 15 and CREM exon 7. (B) This result was further confirmed by RT-PCR, with an alternative fusion transcript being identified secondary to alternative splicing. DNA breakpoints in EWSR1 intron 15 and CREM intron 6 was also identified by long range DNA PCR. (C) CREM exons 7 and 8, included in the fusion transcript, showed up-regulated expression compared to the CREM 5′ exons and other control samples with EWSR1 fusions (Ewing sarcoma, sclerosing epithelioid fibrosarcoma, myoepithelial tumor, hyalinizing clear cell carcinoma of salivary gland) or not (other 1–4). Among the control samples, one Ewing sarcoma with EWSR1-ERG fusion and one myoepithelial tumor with EWSR1-PBX3 fusion also showed elevated downstream CREM exons expression, possibly due to preferential expression of ICER isoform. CREM exons 7 and 8 encode the bZIP domain responsible for dimerization and DNA binding. pKID mediates interaction with the coactivator CBP. (D) Break-apart FISH assay showed CREM rearrangement with telomeric deletion.

Figure 2. Histologic findings of EWSR1-CREM positive index case 1

(A,B) The pelvic tumor showed circumscribed borders and a lobulated growth, composed of alternating hypo and hyper-cellular areas. (C) The hypocellular myxoid areas showed uniform round to ovoid cells arranged in cord-like or reticular patterns. Scattered amianthoid fibers were seen in both the primary (B) and recurrent tumors (C). (D) Cellular areas showed densely packed cells with indistinct cell border and palely eosinophilic cytoplasm.

Figure 3. Morphologic spectrum of intra-cranial tumors

(A–C) Case 2 showed a myxoid neoplasm with a lobulated growth and focal peripheral lymphoid cuff, being composed of loosely arranged uniform round to ovoid cells with occasional amianthoid fibers. (D) Case 3 similarly showed a predominant myxoid stroma with reticular growth pattern. (E–I). Case 4 had a lobulated pattern, with hypocellular myxoid areas exhibiting reticular to adenoid architecture, while hypercellular regions showed whorl-like growth. Immunohistochemically, the cells were weakly positive for EMA (H) and focal strongly positive for desmin (I). (J–L) Case 5 revealed a pushing border into adjacent brain tissues; with solid growth of uniform round cells with occasional cytoplasmic vacuoles. The tumor also showed a myxoid stroma with reticular growth pattern.