Novel ZC3H7B-BCOR, MEAF6-PHF1, and EPC1-PHF1 fusions in ossifying fibromyxoid tumors--molecular characterization shows genetic overlap with endometrial stromal sarcoma

Abstract

PHF1 gene rearrangements have been recently described in around 50% of ossifying fibromyxoid tumors (OFMT) including benign and malignant cases, with a small subset showing EP400-PHF1 fusions. In the remaining cases no alternative gene fusions have been identified. PHF1-negative OFMT, especially if lacking S100 protein staining or peripheral ossification, are difficult to diagnose and distinguish from other soft tissue mimics. In seeking more comprehensive molecular characterization, we investigated a large cohort of 39 OFMT of various anatomic sites, immunoprofiles and grades of malignancy. Tumors were screened for PHF1 and EP400 rearrangements by FISH. RNA sequencing was performed in two index cases (OFMT1, OFMT3), negative for EP400-PHF1 fusions, followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired-end RNA-seq data. Two novel fusions were identified ZC3H7B-BCOR in OFMT1 and MEAF6-PHF1 in OFMT3. After being validated by FISH and RT-PCR, these abnormalities were screened on the remaining cases. With these additional gene fusions, 33/39 (85%) of OFMTs demonstrated recurrent gene rearrangements, which can be used as molecular markers in challenging cases. The most common abnormality is PHF1 gene rearrangement (80%), being present in benign, atypical and malignant lesions, with fusion to EP400 in 44% of cases. ZC3H7B-BCOR and MEAF6-PHF1 fusions occurred predominantly in S100 protein-negative and malignant OFMT. As similar gene fusions were reported in endometrial stromal sarcomas, we screened for potential gene abnormalities in JAZF1 and EPC1 by FISH and found two additional cases with EPC1-PHF1 fusions.

Figure 1

Morphologic spectrum of OFMT harboring different fusion transcript types. Low power reveals a thick peripheral rim of lamellar bone accompanied by an incomplete cartilaginous cap (A), other areas showed densely hyalinized nodules, lacking mineralization (B), while central areas showed the cellular component admixed with lamellar bone (C) (A–C, OFMT1, ZC3H7B-BCOR fusion); (D) Peripheral ossification in OFMT3 showing MEAF6-PHF1; (E) benign OFMT showing partly ossified partly hyalinized pseudocapsule (OFMT7, EP400-PHF1 fusion); (F) a subset of malignant OFMT showed lesional cells embedded within osteoid matrix, reminiscent of osteosarcoma (OFMT3, MEAF6-PHF1); or (G) thick fibrous bands separating the tumor into broad compartments (OFMT14, EP400-PHF1); (H) Benign OFMT with classic cord-like arrangement separated by a dense collagenous stroma (OFMT26, PHF1 rearranged); (I) epithelioid phenotype with more abundant eosinophilic cytoplasm (OFMT15, EP400-PHF1); or (J) a distinctive rhabdoid appearance (OFMT7, EP400-PHF1); (K) malignant OFMT showing a biphasic appearance composed of a benign hypocellular component associated with a conspicuous fibrous stroma, in abrupt transition to a (L) malignant cellular component with high mitotic activity (OFMT21, EP400-PHF1); (M) rare cases of malignant OFMT showed a more spindled/fusiform appearance, arranged in short intersecting fascicles (OFMT20, EP400-PHF1); (N) Most malignant OFMT showed increased cellularity and mitotic activity (OFMT1, ZC3H7B-BCOR); (O) with a loose extracellular stroma and focally very high mitotic activity (5 mitoses, highlighted with arrows) (OFMT33, PHF1 rearranged). (P) The pattern of S100 protein reactivity in malignant OFMT was typically focal (OFMT33, PHF1 rearranged).

Figure 2

ZC3H7B-BCOR gene fusion in malignant ossifying fibromyxoid tumor (OFMT1). (A) Schematic representation of the ZC3H7B-BCOR fusion indicating the loci that are joint together; ZC3H7B exon 10 being fused to BCOR exon 7; (B) RNA reads covering the fusion junction were isolated independent to FusionSeq analysis work flow, supporting the ZC3H7B-BCOR fusion candidate; (C) Experimental validation of the fusion by RT-PCR shows the junction sequence between exon 10 of ZC3H7B and exon 7 of BCOR; (D) Long range DNA PCR showing the fusion of intron 10 of ZC3H7B to the intron 6 of BCOR.

Figure 3

MEAF6-PHF1 gene fusion in malignant ossifying fibromyxoid tumor (OFMT3). (A) Schematic representation of the MEAF6-PHF1 fusion indicating the loci that are joint together; MEAF6 exon 5 being fused to PHF1 exon 2; (B) RNA reads covering the fusion junction were isolated independent to FusionSeq analysis work flow, supporting this fusion candidate; (C) Experimental validation of the fusion by RT-PCR shows the junction sequence between exon 5 of MEAF6 to exon 2 of PHF1.

Figure 4

FISH validation of OFMT-related gene rearrangements. (A) Fusion assay with BCOR (green, telomeric) and ZC3H7B (red, centromeric) showing one yellow fused signal (OFMT1, male patient, only one BCOR allele on Xp11); (B) Break-apart assay showing a split MEAF6 signal (OFMT4; red centromeric, green telomeric); (C) Unbalanced EP400 gene rearrangement, showing break-apart signal with deletion of telomeric (green) part (OFMT14; red, centromeric); (D) PHF1 break apart signal (OFMT14; red centromeric, green, telomeric).