TAF15::NR4A3 gene fusion identifies a morphologically distinct subset of extraskeletal myxoid chondrosarcoma mimicking myoepithelial tumors

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain differentiation predominantly arising in deep soft tissue. Its conventional morphologic appearance manifests as a relatively well‐circumscribed, multilobular tumor composed of uniform short spindle‐to‐ovoid primitive mesenchymal cells with deeply eosinophilic cytoplasm arranged in anastomosing cords within abundant myxoid matrix. The genetic hallmark of EMC has long been considered to be pathognomonic gene rearrangements involving NR4A3, which when fused to TAF15, often have high‐grade morphology with increased cellularity, moderate to severe cytologic atypia, and rhabdoid cytomorphology. Herein, we describe two cases of EMC with TAF15::NR4A3 fusion that appear morphologically distinct from both conventional and high‐grade EMC. Both cases had an unusual biphasic appearance and showed diffuse positivity for p63, mimicking myoepithelial tumors. DNA methylation profiling demonstrated that both cases clearly cluster with EMC, indicating that they most likely represent morphologically distinct variants of EMC. The clinical significance and prognostic impact of this morphologic variance remains to be determined. Molecular testing, including DNA methylation profiling, can help to confirm the diagnosis and avoid confusion with mimics; it adds another layer of data to support expanding the morphologic spectrum of EMC.


| INTRODUCTION
Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain differentiation, predominantly arising in soft tissue and rarely occurring in bone. 1 Early descriptions of EMC include a case of a "chordoid tumor" first described by Stewart in 1948, 2 as well as a series of cases originally believed to be de novo chondrosarcomas arising in extraskeletal soft tissue by Stout and Verner in 1953. 3 EMC was officially recognized as a distinct clinicopathologic entity in 1972 by Enzinger and Shiraki,4 who coined the term based on the resemblance to embryonic cartilage. Despite early descriptions and persisting nomenclature, EMC is considered distinct from conventional chondrosarcoma with myxoid matrix, 5 and the phenotype of the neoplastic cells is not chondrogenic.
EMC typically arises in the deep soft tissue of the proximal extremities (80%) with the thigh being the most common site. While it can occur over a wide age range, middle-aged adults are most frequently affected with a median age of 50 years. The clinical course is often protracted with a tendency for late local recurrence (48%) and metastasis (46%), particularly to the lungs, followed by soft tissue, lymph nodes, bone, and brain. 6 A genetic hallmark of EMC has long been considered the pathognomonic rearrangements involving NR4A3 (9q22. 33), 7 which are identified in more than 90% of EMC. 8 Although EWSR1 (22q12) is the most common fusion partner for NR4A3, genetic heterogeneity exists, with alternative fusion partners described including TAF15 (17q12), 9 FUS (16p11.2), 10 TCF12 (15q21), 11 TGF (3q12.2), 12 SMARCA2 (9p24.3), 13 LSM14A (19q13.11) 14 and HSPA8 (11q24.1). 15 In 75%-80% of cases of EMC, NR4A3 is fused with EWSR1, 7,16 and this is accompanied by a conventional morphologic appearance, manifesting as a relatively well-circumscribed, multilobular tumor composed of uniform short spindle-to-ovoid primitive mesenchymal cells with a modest amount of deeply eosinophilic to vacuolated cytoplasm arranged in anastomosing cords, forming a complex interconnecting reticular network enmeshed in an abundant basophilic myxoid matrix. Less frequently, NR4A3 may be fused to TAF15 (16%-27%), 7,16 and the majority of these cases have been described as having high-grade morphology with increased cellularity, mitotic activity, and cytologic atypia with limited myxoid matrix and rhabdoid cytomorphology in approximately half of the cases. 7 Expanding on prior descriptions, we herein describe two cases of EMC with confirmed TAF15::NR4A3 fusion that appeared morphologically distinct from both conventional and high-grade EMC. Both tumors further had a distinct biphasic appearance and strong immunohistochemical expression of p63, mimicking myoepithelial tumors. Both tumors underwent DNA methylation profiling using the Infinium HumanMethylation EPIC array platform as previously described. 17 Probes associated with known SNPs, non-CpGs, and sex chromosomes were not taken into account for the evaluation. Moreover, samples with a mean detection p > 0.02 were discarded. Uniform manifold approximation and projection (UMAP) was performed on the results of a principal component analysis (50 PCs) calculated via the singular value decomposition of the beta methylation matrix.

| MATERIALS AND METHODS
The R-package used for generating the graph can be found at Copy number variations were inferred from the Infinium Human-Methylation EPIC Array platform using the R-package "conumee." The settings for the copy number variation inference were as follows: a minimum number of probes per bin equal to 25; minimum bin size equal to 100 000 bp. Immunohistochemical stains showed that the tumor cells were strongly positive for p63 ( Figure 1F, inset), while they were essentially negative for ER, PR, S100 protein, SOX10, EMA, CK AE1/AE3, CK Limited follow-up at 7 months showed no evidence of recurrence or metastatic disease.

| Case 2
A 49-year-old woman with a history of trauma to her right back after sustaining a fall 10 years prior presented with a palpable mass, arising in the same area, that had been present for many years and was gradually increasing in size. A targeted ultrasound revealed a soft tissue mass involving the right flank, and follow-up imaging confirmed a solid, enhancing mass in the subcutaneous tissue of the right lower  Immunohistochemical stains showed that the tumor cells were strongly positive for p63 ( Figure 3F, inset), while they were essentially negative for CK AE1/AE3, CK 5/6, EMA, S100 protein, SMA, desmin,   Despite initial assertions, essentially no gene fusion has proven to be entirely tumor-specific; rearrangements involving NR4A3, once thought to be pathognomonic for EMC, are no exception. In addition to EMC, NR4A3 is specifically and consistently upregulated in salivary acinic cell carcinoma, which also harbors NR4A3 rearrangements. 20 This finding has made nuclear NR4A3 immunostaining a highly sensitive (92%-98%) and specific (97%-100%) marker for acinic cell carcinoma of salivary gland origin. 20 pleomorphism, marked mitotic activity, and more vascular stroma with minimal myxoid matrix. In the largest series to date, all cases with moderate to high cellularity have also been reported to demonstrate moderate to severe cytologic atypia. 7 Some cases of high-grade EMC appear to transition from a conventional EMC or transform with tumor recurrence, even as late as 14 years after primary resection. 25,26 Between 4 and 29% of EMC show cellular areas devoid of myxoid matrix 6,7 ; however, a lobular architecture is maintained, and the underlying conventional low-grade component is usually readily identifiable. Additional cases of conventional EMC juxtaposed to a high-grade spindle cell sarcoma have been reported, which may represent dedifferentiation. 5,27 Although the two cases in this study had limited to no myxoid matrix, similar to high-grade EMC, the morphologic features were distinctly different from what has been described as either conventional or high-grade EMC. Both cases lacked severe cytologic atypia and rhabdoid cells, which are typically associated with high-grade EMC.
Likewise, no areas with an underlying conventional component were Myoepithelial tumors are morphologically and clinically similar to EMC as they are usually well-circumscribed with a lobular growth pattern, frequently have myxoid stroma, and often arise in the limbs or limb girdles of adults in their 3rd to 5th decade. Myoepithelial tumors are often positive for p63, but they are also frequently positive for other myoepithelial markers including CK AE1/AE3, SMA, S100 protein, GFAP, and calponin-all of which were negative in our cases.
While conventional EMC can show infrequent or focal positivity for p63, 28 strong and diffuse expression is rare but has previously been reported in one case of cellular EMC with TAF15::NR4A3 fusion. 29 Genetic similarities between EMC and myoepithelial tumors also exist as both entities can have gene rearrangements involving EWSR1; however, gene rearrangements involving NR4A3 are not associated with myoepithelial tumors. 29 Tumors with morphologic features of myoepithelial tumors that lack the expected immunophenotype have also recently been described under the terminology "myoepitheliomalike" tumor. 30,31 Within soft tissue, these lesions have been described in the hands and feet, with frequent immunoreactivity for EMA and CD34, characterized molecularly by gene rearrangement of OGT with FOXO3 or FOXO1. [31][32][33] While the two cases presented here have some morphologic and immunophenotypic overlap with these myoepithelioma-like soft tissue tumors, the current cases are anatomically more proximally located, lack CD34 and EMA reactivity, and harbor a different gene rearrangement.
Prior to DNA methylation profiling, the most appropriate classification for these two tumors was not evident. The unusual morphologic features with a biphasic appearance and strong expression of p63 raised the possibility of myoepithelial or myoepithelial-like tumors. However, DNA methylation profiling demonstrated that both tumors clearly clustered with EMC, indicating that they most likely represent distinct morphologic variants of EMC. While NR4A3 fusions have now been described in other mesenchymal neoplasms, the presence of TAF15::NR4A3 gene fusion in each case still supported classification as EMC. Furthermore, it is interesting to note that both cases, along with the EMC group as a whole, clustered tightly together, separate from conventional chondrosarcoma, including those with and without IDH mutations, as has been previously described. 17 These results underscore that EMC truly represents a distinct sarcoma separate from conventional chondrosarcoma with a wider morphologic spectrum than previously recognized. To the best of our knowledge, myoepithelial and myoepithelial-like tumors have not yet been systematically studied through methylation profiling, and it remains to be determined how they cluster with respect to EMC.
The treatment of choice for conventional EMC remains complete surgical resection with or without radiation. For patients with unresectable recurrent or metastatic disease, chemotherapeutic agents may be tried often with poor response. Anthracycline-based chemotherapy is the standard front-line regimen, and trabectedin 34 or pazopanib 35 may be used as second-line. Several cases with EWSR1:: NR4A3 fusion have shown a response with the tyrosine kinase inhibitor sunitinib; however, patients with TAF15 fusion continued to progress. 36 This result mirrors results by Paioli et al. who showed that patients with EWSR1::NR4A3 trended toward better disease-free survival. 16 The true prognostic impact of both the unusual morphologic features and fusion type (e.g., TAF15::NR4A3) remains to be determined.
Most cases of EMC have a propensity for late recurrence regardless of fusion type, indicating the importance of long-term clinical followup in these cases. As these cases continue to be followed, and more cases with variant morphology are identified, the clinical course of these tumors will become more evident. Molecular testing, including DNA methylation profiling, can help to confirm the diagnosis and avoid confusion with mimics as the morphologic spectrum of EMC expands.

DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are included within the article.