Expanding the molecular signature of ossifying fibromyxoid tumors with two novel gene fusions: CREBBP-BCORL1 and KDM2A-WWTR1

Abstract

Ossifying fibromyxoid tumor (OFMT) is an uncommon mesenchymal neoplasm of uncertain differentiation and intermediate malignant potential. Recurrent gene fusions involving either PHF1 or BCOR have been found in 85% of OFMT, including typical and malignant examples. As a subset of OFMT still lack known genetic abnormalities, we identified two OFMTs negative for PHF1 and BCOR rearrangements, which were subjected to transcriptome analysis for fusion discovery. The RNA sequencing found a novel CREBBP-BCORL1 fusion candidate in an axillary mass of a 51 year-old male and a KDM2A-WWTR1 in a thigh mass of a 36 year-old male. The gene fusions were validated by RT-PCR and FISH in the index cases and then screened by FISH on 4 additional OFMTs lacking known fusions. An identical CREBBP-BCORL1 fusion was found in an elbow tumor from a 30 year-old male. Both OFMTs with CREBBP-BCORL1 fusions had areas of typical OFMT morphology, exhibiting uniform round to epithelioid cells arranged in cords or nesting pattern in a fibromyxoid stroma. The OFMT with KDM2A-WWTR1 fusion involved dermis and superficial subcutis, being composed of ovoid cells in a fibromyxoid background with hyalinized giant rosettes. The S100 immunoreactivity ranged from very focal to absent. Similar to other known fusion genes in OFMT, BCORL1, CREBBP and KDM2A are also involved in histone modification. In summary, we expand the spectrum of molecular abnormalities in OFMT with 2 novel fusions, CREBBP-BCORL1 and KDM2A-WWTR1, further implicating the epigenetic deregulation as the leading pathogenetic mechanism in OFMT. © 2016 Wiley Periodicals, Inc.

Figure 1. CREBBP-BCORL1 fusion in OFMT

RNA sequencing fusion discovery showed fusion junction reads mapped to the exon 30 of CREBBP (16p13.3) and exon 4 of BCORL1 (Xq26.1); results further confirmed by RT-PCR and Sanger sequencing (A,B). The predicted CREBBP chimeric protein reveals the histone acetyltransferase (HAT) domain being retained in the fusion and joined to the C-terminal part of BCORL1 (B). FISH analyses showed CREBBP and BCORL1 break-apart signals (green, telomeric and red, centromeric, arrows, C). BCORL1 on Xq26 shows split-apart of the only allele in this male patient. High BCORL1 mRNA expression (distal to the exon 4 breakpoint) was noted in the CREBBP-BCORL1 positive OFMT, compared to OFMTs with other gene fusions, including MEAF6-PHF1, EP400-PHF1, and ZC3H7B-BCOR (D,E). Additionally full cDNA BCORL1 overexpression was also identified in round cell sarcomas with BCOR-gene abnormalities (BCOR-MAML3 or BCOR ITD) (D).

Figure 2. KDM2A-WWTR1 fusion in OFMT

Schematic diagram and fusion FISH assay demonstrate the 5′ KDM2A (centromeric, red) being fused to the inverted 3′ WWTR1 (centromeric, yellow) (A). RT-PCR confirms the KDM2A-WWTR1 in-frame transcript (B). The chimeric protein retains the entire WWTR1 coding region.

Figure 3. Pathologic findings of OFMTs with CREBBP-BCORL1 (A–F) and KDM2A-WWTR1 fusions (G–I)

Case#1 was composed of uniform evenly-spaced small round cells arranged in cord-like structures in a fibromyxoid to hyalinized stroma (A,B), which based on the increased cellularity and mitotic activity was classified as malignant (arrows, C). Case#3 showed alternating collagenous and myxoid areas with focal microcystic change (D), with bland round cells with uniform nuclei and scant cytoplasm (E) arranged in cord-like to reticular patterns (F). The KDM2A-WWTR1 positive OFMT#2 was located within dermis (G) and superficial subcutis. It was composed of uniform ovoid tumor cells set in a loose fibromyxoid background (H). The tumor showed distinctive giant collagen rosettes throughout (I).