EWSR1-PBX3: a novel gene fusion in myoepithelial tumors

Abstract

The genetics of myoepithelial tumors (ME) of soft tissue and bone have recently been investigated, with EWSR1-related gene fusions being seen in approximately half of the tumors. The fusion partners of EWSR1 so far described include POU5F1, PBX1, ZNF444 and, in a rare case, ATF1. We investigated by RNA sequencing an index case of EWSR1-rearranged ME of the tibia, lacking a known fusion partner, and identified a novel EWSR1-PBX3 fusion. The fusion was further validated by reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization (FISH). To evaluate if this is a recurrent event, an additional cohort of 22 EWSR1-rearranged ME cases lacking a fusion partner were screened by FISH for abnormalities in PBX3 gene. Thus, two additional cases were identified showing an EWSR1-PBX3 gene fusion. One of them was also intraosseous involving the ankle, while the other occurred in the soft tissue of the index finger. The morphology of the EWSR1-PBX3 fusion positive cases showed similar findings, with nests or sheets of epithelioid to spindle cells in a partially myxoid to collagenous matrix. All three cases showed expression of S100 and EMA by immunohistochemistry. In summary, we report a novel EWSR1-PBX3 gene fusion in a small subset of ME, thereby expanding the spectrum of EWSR1-related gene fusions seen in these tumors. This gene fusion seems to occur preferentially in skeletal ME, with two of the three study cases occurring in intraosseous locations.

Figure 1

Imaging for index case ME1 (A) Sagittal fat-suppressed T2-weighted MR image of the primary 1.4 cm lesion showing lesion with hyperintense signal, located in the anterior subcortical region of proximal tibial metaphysis. No surrounding marrow edema pattern or soft tissue extension is evident. (B) Axial and (C) coronal CT images without intravenous contrast obtained 44 months later demonstrate increase in size (3.8 cm) of the lytic lesion. The lesion is multilobulated, and contains multiple internal septations, a thin sclerotic margin and no calcified matrix. (D) Lateral radiograph obtained 3 months later shows the peripheral sclerotic margin and septations. Axial (E) T1-weighted and (F) post-gadolinium fat-suppressed T1-weighted MR images obtained 33 months later show the low-signal, non-enhancing surgical cement (*), as well as a small focus of recurrent tumor (arrow) that has developed along the anterolateral margin of cement, with intermediate T1 signal and avid enhancement.

Figure 2

ME tumor involving the tibial metaphysis in a 26 year-old male (ME1). Low power image from the first curettings showing epithelioid cells in a myxoid stroma (A, 200x), while other areas displaying spindle cells in short fascicles. (B, 100x) Recurrent lesion showing epithelioid cells in a partially sclerotic stroma (C, 100x); FISH break-apart assay for PBX3 gene showing split signal indicating rearrangement (D).

Figure 3

EWSR1-PBX3 gene fusion in ME tumor (ME1). (A) Schematic representation of the EWSR1-PBX3 fusion indicating the chromosomal loci joint together; EWSR1 exon 8 being fused to PBX3 exon 5; (B) RNA reads covering the fusion junction were isolated independent to FusionSeq analysis work flow, supporting the EWSR1-PBX3 fusion candidate; (C) RT-PCR experimental validation of the fusion shows the EWSR1 exon 8 fused to PBX3 exon 5; (D) DNA PCR further confirming the genomic breakpoint, showing the fusion of intron 9 of EWSR1 to the intron 4 of PBX3.

Figure 4

Pathologic spectrum of EWSR1-PBX3 fusion positive ME tumors. ME tumor involving the fibula in a 16 year-old female (ME2) showing nests of ovoid cells (A, 100x), with focally more epithelioid cells having clear cytoplasm (B, 400x); ME lesion involving the index finger soft tissue in a 53 year-old female (ME3) showing sheets of monomorphic cells (C, 100x), which at higher power reveal ovoid or epithelioid cells with prominent nucleoli in a loose matrix (D, 400x).