Implementation of Copy Number Variations-Based Diagnostics in Morphologically Challenging EWSR1/FUS::NFATC2 Neoplasms of the Bone and Soft Tissue

In the last decade, new tumor entities have been described, including EWSR1/FUS::NFATC2-rearranged neoplasms of different biologic behavior. To gain further insights into the behavior of these tumors, we analyzed a spectrum of EWSR1/FUS::NFATC2-rearranged neoplasms and discuss their key diagnostic and molecular features in relation to their prognosis. We report five patients with EWSR1/FUS::NFATC2-rearranged neoplasms, including one simple bone cyst (SBC), two complex cystic bone lesions lacking morphological characteristics of SBC, and two sarcomas. In three cases, fluorescence in situ hybridization (FISH) and in all cases copy number variation (CNV) profiling and fusion analyses were performed. All patients were male, three cystic lesions occurred in children (aged 10, 14, and 17 years), and two sarcomas in adults (69 and 39 years). Fusion analysis revealed two FUS::NFATC2 rearrangements in two cystic lesions and three EWSR1::NFATC2 rearrangements in one complex cystic lesion and two sarcomas. EWSR1 FISH revealed tumor cells with break-apart signal without amplification in one complex cystic lesion and EWSR1 amplification in both sarcomas was documented. CNV analysis showed simple karyotypes in all cystic lesions, while more complex karyotypes were found in NFATC2-rearranged sarcomas. Our study supports and expands previously reported molecular findings of EWSR1/FUS::NFATC2-rearranged neoplasms. The study highlights the importance of combining radiology and morphologic features with molecular aberrations. The use of additional molecular methods, such as CNV and FISH in the routine diagnostic workup, can be crucial in providing a correct diagnosis and avoiding overtreatment.


Introduction
The WHO classification of primary bone and soft tissue neoplasms has been constantly refined over the past two decades. The modifications of the classification scheme have been significantly accelerated by the incorporation of genetic aberrations identified by next generation sequencing (NGS) technologies. In the current fifth edition of the WHO book on Soft Tissue and Bone Tumors, the molecular signature of tumors has been incorporated into Int. J. Mol. Sci. 2022, 23, 16196 2 of 12 their definition, which underscores the importance of molecular diagnostics in the routine work-up of soft tissue and bone neoplasms [1].

Results
Clinical, immunohistochemical, and molecular data are summarized in Table 1. Radiological features are outlined in Table 2. Legend: m-male; NA-not available; NP-not performed; NSD-no sign of disease. A 17-year-old male patient was admitted with pain due to an acute fracture after slipping while walking.. Radiographs revealed within the left proximal femoral diaphysis a 3.2 cm large geographic lucent lesion with a narrow zone of transition and thin septations ( Figure 1A-H). The lesion was curetted and stabilized with internal fixation. Histologically, the tumor was cystic, and the cyst walls were moderately cellular and composed of scattered bland spindle to ovoid cells admixed with scattered foamy histiocytes, osteoclast-like giant cells, and unmineralized fibrin-like collagen (Figure 2A-D). Molecular analysis showed a FUS::NFATC2 fusion ( Figure 3A) and a simple/normal karyotype (Supplementary Figure  S1A). During 12 months of follow-up, the patient was without symptoms and no local recurrence.

Complex Cystic Bone Lesions (Cases #2 & 3)
Two male patients, 10 and 14 years old, presented with pain caused by spontaneous non-traumatic, comminuted, pathologic fractures that involved the right proximal femur and the right proximal humerus diaphysis, respectively. Both lesions were wellcircumscribed, lytic, and expansile with endosteal scalloping. There was no cortical transgression. The tumor in the humerus had thin septations ( Figure 4A-D and Figure 5A-D). The femoral lesion was 2.9 cm large, and the humeral lesion measured 6.7 cm. Both lesions were curetted, and internal fixation was placed.
Histologically, the cyst walls of both tumors were thick and composed of a bland proliferation of spindle and ovoid cells that have vesicular nuclei and pale eosinophilic cytoplasm ( Figure 4E-H and Figure 5E-H). Foamy histiocytes were scattered through the neoplasms. No pleomorphism and only scattered mitotic figures of normal configuration were present. Immunohistochemistry demonstrated that the lesional cells were diffuse positivity for CD99 ( Figure 5H), with multifocal staining for EMA and focal staining for SATB2. The stain for NKX2.2 was negative. Fusion analysis revealed FUS::NFATC2 (case #2; Figure 3B) and EWSR1::NFATC2 (case #3) rearrangements. FISH using a EWSR1 breakapart probe revealed a significant number of cells with break-apart (split) signal (>20% of lesional cells); however, no EWSR1 amplification was present ( Figure 5H inset). CNV analysis showed a simple karyotype without any copy number alterations (Supplementary Figure S1B,C).
The femoral lesion was resected en bloc and reconstructed with a prosthesis. Both patients are in close follow-up, and no recurrences have developed after 12 and 10 months of follow-up. In case #3, follow-up radiographs show reactive cortical thickening and bone remodeling of the defect ( Figure 5D). logically, the tumor was cystic, and the cyst walls were moderately cellular and composed of scattered bland spindle to ovoid cells admixed with scattered foamy histiocytes, osteoclast-like giant cells, and unmineralized fibrin-like collagen (Figure 2A-D). Molecular analysis showed a FUS::NFATC2 fusion ( Figure 3A) and a simple/normal karyotype (Supplementary Figure S1A). During 12 months of follow-up, the patient was without symptoms and no local recurrence.   A 69-year-old male patient complained of a slowly growing mass in the right lower leg. MRI showed a well-demarcated intramuscular lesion in the distal fibularis brevis muscle with peripheral contrast enhancement ( Figure 6A-D). The tumor was resected with wide margins. Microscopically, the tumor was heterogeneous in appearance. In the hypocellular areas, it was composed of a myxohyaline or cartilaginous stroma-rich component that contained monomorphic ovoid to epithelioid tumor cells arranged in interconnecting cords and nests adjacent to a hypercellular component. The tumor cells in the hypercellular foci consisted of atypical spindle and round cells arranged in packed cords, nests, and solid sheets ( Figure 6E-H). Focally, metaplastic bone was present. Mitotic activity was limited (<5 mitosis/10 HPF), and areas of necrosis were not present. Immunohistochemistry showed cytoplasmic and membranous staining with CD99, and EMA was focally positive. NGS revealed EWSR1::NFATC2 fusion ( Figure 3C). FISH showed break apart of       non-traumatic, comminuted, pathologic fractures that involved the right proximal femur and the right proximal humerus diaphysis, respectively. Both lesions were well-circumscribed, lytic, and expansile with endosteal scalloping. There was no cortical transgression. The tumor in the humerus had thin septations ( Figures 4A-D and 5A-D). The femoral lesion was 2.9 cm large, and the humeral lesion measured 6.7 cm. Both lesions were curetted, and internal fixation was placed.

Discussion
Numerous investigations have documented the increasing promiscuity of gene fusions in bone and soft tissue neoplasms. Accordingly, a precise accurate diagnosis requires correlation of the radiology, morphology, immunohistochemical, and genetic findings.
In our study, we analyzed a biological spectrum of bone and soft tissue EWSR1/FUS::NFATC2-rearranged neoplasms. Clinical findings were similar to those previously reported [5,8,9]. SBC and related complex cystic lesions occurred in children in the first two decades, and round-cell sarcomas developed in adults. All tumors occurred in males and ranged in size from 2 to 7.6 cm. In three cases, the long bones were affected. The EWSR1::NFATC2 bone and soft tissue sarcomas presented as slowly growing masses. This phenomenon is described in several cases of EWSR1/FUS::NFATC2-rearranged sarcomas in which symptoms were present for years before diagnosis, suggesting that a preexisting low-grade component may have undergone biological progression in a subset of patients [8,12].
The histological appearance of the complex cystic bone lesions seems different from that of the wall of a "classic" SBC. In our two complex bone cysts, cellular areas of the monotonous spindle to ovoid cells admixed with abundant foamy macrophages were

Discussion
Numerous investigations have documented the increasing promiscuity of gene fusions in bone and soft tissue neoplasms. Accordingly, a precise accurate diagnosis requires correlation of the radiology, morphology, immunohistochemical, and genetic findings.
In our study, we analyzed a biological spectrum of bone and soft tissue EWSR1/FUS::NF ATC2-rearranged neoplasms. Clinical findings were similar to those previously reported [5,8,9]. SBC and related complex cystic lesions occurred in children in the first two decades, and round-cell sarcomas developed in adults. All tumors occurred in males and ranged in size from 2 to 7.6 cm. In three cases, the long bones were affected. The EWSR1::NFATC2 bone and soft tissue sarcomas presented as slowly growing masses. This phenomenon is described in several cases of EWSR1/FUS::NFATC2-rearranged sarcomas in which symptoms were present for years before diagnosis, suggesting that a pre-existing low-grade component may have undergone biological progression in a subset of patients [8,12].
The histological appearance of the complex cystic bone lesions seems different from that of the wall of a "classic" SBC. In our two complex bone cysts, cellular areas of the monotonous spindle to ovoid cells admixed with abundant foamy macrophages were seen. Recently, Pizem et al., however, described two cases with EWSR1::NFATC2 fusion with features of a classic SBC next to more cellular areas with admixed histiocytes [5].
Frequently, prominent foamy histiocytes are found in non-ossifying fibromas; however, these lesions present with characteristic radiologic findings and KRAS mutation. Moreover, in case #5, as previously described by Diaz-Perez et al. [8], a hypocellular bland spindle cell component with bland morphology was found, suggesting a benign precursor lesion in a tumor with a clear morphologic transition into a hypercellular clearly malignant tumor component. Similar findings were described in two tumors showing a less cellular and more myxoid tumor component, juxtaposed to a hypercellular component with depletion of a matrix, indicating a morphologic spectrum or histologic progression from a low-grade to high-grade histology [10]. Immunohistochemistry with CD99 and EMA plays a limited role, as both benign and malignant EWSR1/FUS::NFATC2-rearranged neoplasms can stain positive [5,8]. Diffuse nuclear NKX2.2 staining, however, is observed only in sarcoma and can be useful in a distinction from benign tumors [4,10]. Our complex cystic lesions showed a diffusely positive staining with CD99, a multifocal staining with EMA, and focal staining with SATB2, whereas NKX2.2 was negative.
CNV analysis provides valuable insights into tumor biology. Analyzing chromosomal abnormalities and genomic instability is important in identifying potential prognostic and predictive biomarkers. In our cases of an SBC and two complex cystic lesions, no genomic alterations using CNV analysis and FISH for EWSR1 amplification were identified. On the other hand, FUS::NFATC2-translocated sarcomas exhibited very complex genomic profiles, while in those cases harboring ESWR1::NFATC2 fusion, fewer genomic alterations were found [6,7,13]. The CNV analysis of the low-grade and high-grade components in case #5, interestingly, revealed additional chromosomal changes in the latter component. It is conceivable that the EWSR1/FUS::NFATC2 fusion might be a very early event in tumor initiation, and additional events are required for progression.
Significant differences in morphology, transcriptional profile, and behavior are found when comparing sarcomas harboring EWSR1::NFATC2 and FUS::NFATC2 fusions [14]. The latter is more aggressive and transcriptionally resembles CIC-rearranged sarcomas. Additionally, recurrent fusion gene amplification is almost exclusively detected in the EWSR1::NFATC2-rearranged sarcomas. The other benign or malignant tumor types harboring FUS or EWSR1 translocation show no gene amplification using FISH [8][9][10]. Higher expression levels of fusion transcripts found in EWSR1::NFATC2-rearranged sarcomas could also be an explanation for the malignant behavior [3].

Patients and Tumor Characteristics
Five EWSR1/FUS::NFATC2-rearranged neoplasms, including one simple bone cyst, one soft tissue round cell sarcoma, and two complex cystic bone lesions diagnosed at the D&R Institute of Pathology, Medical University of Graz, from 2018-2022, and one round cell bone sarcoma previously reported in 2019 [8], were analyzed. Institutional ethical approval was obtained from the Ethics Committee of Medical University of Graz. All cases were reviewed by two bone and soft tissue pathologists (I.B. and B.L.-A.).

RNA and DNA Workflow
For each sample, genomic DNA and RNA were extracted from FFPE material (10-12 unstained, 10 µm thick sections). H&E-stained sections from a single representative FFPE block were examined, areas of high tumor content were marked, and microdissection was performed using a needle to enrich for tumor content. RNA and DNA were isolated using the Maxwell RSC FFPE Plus RNA/DNA Purification Kit (AS1440/AS1720, Promega, Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer s instructions. RNA was quantified by the QuantiFluor ONE dsDNA System on the Quantus Fluorometer (both Promega).

Tumor-Associated Copy Number Variation Analysis
50 ng genomic DNA were used to prepare libraries with the NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent (New England Biolabs, Hitchin, UK). To obtain libraries with a length between 250 and 350 bp the products were electrophoresed using the E-Gel SizeSelect 2% Agarosegel and the E-Gel Safe Imager (both Thermo Fischer Scientific). A qPCR of the finished libraries was then made with the CFX C1000 Touch Thermal Cycler and CFX96 Real-Time System (Bio-Rad). Sequencing was done on the Ion S5 System (Instrument: Ion S5 Sequenzer and Server) and Ion Chef Instrument (Thermo Fischer Scientific). About 5,000,000 reads were taken into calculation, visualized and manually evaluated. Ploidy of the sample was calculated via a reference sample of the opposite gender according to the patients.

Conclusions
EWSR1/FUS::NFATC2 rearrangements are promiscuous gene fusions found in both benign and malignant bone and soft tissue tumors. The presence of a specific fusion does not determine the diagnosis, clinical behavior, or response to therapy. Therefore, a thorough correlation of radiology, morphology, immunohistochemical profile, and molecular findings is indispensable in rendering the correct diagnosis. It seems that SBCs can present with a broad morphological spectrum ranging from thin-to thick-walled tumors that are hypo to hypercellular with or without numerous foamy histiocytes. Our results indicate that the presence of EWSR1 gene amplification or gene copy number changes is found only in biologically aggressive neoplasms. In challenging cases, tumors with EWSR1/FUS::NFATC2 rearrangements can undergo CNV and FISH analysis to help determine the biological potential of the neoplasm and the appropriate treatment and follow-up.