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INTRODUCTION The SS18::NEDD4 fusion gene has been recently identified in only one case of primary renal synovial sarcoma (SS) by Argani et al. Although not yet fully characterized, this gene fusion was reported in a variant of SS, an entity most notably characterized by the t(X;18)(p11;q11) translocation resulting in SS18 gene rearrangements involving fusion partners SSX1 in two‐thirds of SS, SSX2 in one‐third, and rarely SSX4 . , , , The detection of SS18::SSX1/2/ 4 fusion gene is considered the gold standard in the diagnosis of SS. However, the increasing use of next‐generation sequencing (NGS), in modern clinical molecular laboratories has enabled the discovery of many novel gene fusions well as molecular pleiotropy in both solid organ and hematologic malignancies. Consequently, the detection of SS18 rearrangements in entities other than SS has been reported. Specifically, SS18 rearrangements involving the MEF2C and MEF2D partner genes, resulting in MEF2C::SS18 and MEF2D:SS18 gene fusions, have been identified in microsecretory adenocarcinoma and the B‐cell acute lymphoblastic leukemia, respectively. However, both entities, microsecretory adenocarcinoma of the salivary gland, and acute B‐cell lymphoblastic leukemia, arise from different cellular lineages and display unique histomorphology and clinical courses. , Furthermore, the translocation resulting in CRTC::SS18 has been identified in an undifferentiated small round cell sarcoma with aberrant ALK expression. Other undifferentiated round cell sarcomas with novel SS18‐POU5F1 have also been described previously. The heterogeneity in histomorphology of tumors harboring SS18 rearrangements highlights the potential cell‐specific variability of perturbations in BAF regulation, a chromatin remodeling complex that functions to maintain balance in both gene activation and repression. Herein, we describe an aggressively behaving malignant cutaneous spindled and epithelioid sarcoma harboring an SS18::NEDD4 gene fusion in a 60‐year‐old female. To date, the SS18::NEDD4 fusion has only been identified in one case of primary renal SS. To the best of our knowledge, this index case represents the first report of SS18::NEDD4 gene fusion in an unclassified sarcoma, other than SS. 

MATERIALS AND METHODS 2.1 Immunohistochemistry Immunohistochemical stains using the following commercially available antibodies were performed using the Leica Bond III autostainers (Leica Biosystems): AE1/E3 (Dako, AE1&AE3, 1:1200), CK7 (Dako, OV‐TL‐12/30, 1:600), CK20 (Dako, Ks20.8, 1:200), CAM 5.2 (BD, CAM5.2, 1:80), vimentin (Dako, V9, 1:1800), BCL‐2 (Dako, 124, 1:1200), smooth muscle actin (Dako, 1A4, 1:600), P63 (Biocare, 4A4, 1:300), Desmin (Dako, D33, 1:200), S‐100 (Dako, R poly, 1:2000), INI1 (BD, BAF47, 1:2000), CD56 (Dako, 123C3, 1:100), TLE1 (BioSB, 1F5, 1:50), P40 (Biocare, BC28, 1:150), P53 (Dako, DO‐7, 1:1000), CMYC (Cell Marque, EP121[Y69], 1:100), ERG (BioCare M, 9FY, 1:50), CD34 (Cell Marque, QBEnd/10, 1:400), CD31 (Dako, JC 70A, 1:400), H‐caldesmon (Biogenex, clone‐h‐CD, 1:500), smooth muscle myosin heavy chain, SMMS (Cell Marque, clone‐SMMS‐1, 1:800), and myogenin (Cell Marque/F5D, 1:200). All protocols were developed by and performed at the OSU Wexner Medical Center Histology Laboratory, 680 Akerman Road, Columbus, OH 43210. All positive and negative controls showed appropriate staining. 2.2 Next‐generation sequencing NGS was performed using the FoundationOne Heme assay (Foundation Medicine, Inc. Cambridge, MA). Total nucleic acid, DNA, and RNA, was extracted from visible tumor in 4‐μm sections of formalin‐fixed paraffin‐embedded tissue. Comprehensive genomic profiling was performed using FoundationOne Heme® (F1H), a clinical‐grade, high‐throughput, hybridization capture‐based NGS assay for targeted sequencing of all exons of 406 genes, and RNA sequencing of 265 genes. F1H methods used have been previously reported in detail. Sequences were analyzed for genomic alterations including short variant alterations, copy number alterations, and gene rearrangements as described. 2.3 Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) analysis was performed on a 4‐μm unstained section of representative formalin‐fixed, paraffin‐embedded tissue using a commercially available LSI break‐apart probe set (Vysis). The LSI SS18 Dual Color, Break Apart Rearrangement Probe consists of a mixture of two FISH DNA probes. The first probe, an ~650 kb probe labeled in SpectrumOrangeTM, extends distally from the SS18 gene. The second probe labeled in SpectrumGreenTM lies 3′ or proximal to the SS18 gene and is approximately 1040 kb in length. Hybridization signals were assessed in 200 interphase nuclei with strong, well‐delineated signals. Only nonoverlapping tumor nuclei with a complete set of signals were scored. The cut‐off level for scoring aberrations was 15% abnormal nuclei for the dual‐color SS18 FISH probe system. 

Immunohistochemistry Immunohistochemical stains using the following commercially available antibodies were performed using the Leica Bond III autostainers (Leica Biosystems): AE1/E3 (Dako, AE1&AE3, 1:1200), CK7 (Dako, OV‐TL‐12/30, 1:600), CK20 (Dako, Ks20.8, 1:200), CAM 5.2 (BD, CAM5.2, 1:80), vimentin (Dako, V9, 1:1800), BCL‐2 (Dako, 124, 1:1200), smooth muscle actin (Dako, 1A4, 1:600), P63 (Biocare, 4A4, 1:300), Desmin (Dako, D33, 1:200), S‐100 (Dako, R poly, 1:2000), INI1 (BD, BAF47, 1:2000), CD56 (Dako, 123C3, 1:100), TLE1 (BioSB, 1F5, 1:50), P40 (Biocare, BC28, 1:150), P53 (Dako, DO‐7, 1:1000), CMYC (Cell Marque, EP121[Y69], 1:100), ERG (BioCare M, 9FY, 1:50), CD34 (Cell Marque, QBEnd/10, 1:400), CD31 (Dako, JC 70A, 1:400), H‐caldesmon (Biogenex, clone‐h‐CD, 1:500), smooth muscle myosin heavy chain, SMMS (Cell Marque, clone‐SMMS‐1, 1:800), and myogenin (Cell Marque/F5D, 1:200). All protocols were developed by and performed at the OSU Wexner Medical Center Histology Laboratory, 680 Akerman Road, Columbus, OH 43210. All positive and negative controls showed appropriate staining. 

Next‐generation sequencing NGS was performed using the FoundationOne Heme assay (Foundation Medicine, Inc. Cambridge, MA). Total nucleic acid, DNA, and RNA, was extracted from visible tumor in 4‐μm sections of formalin‐fixed paraffin‐embedded tissue. Comprehensive genomic profiling was performed using FoundationOne Heme® (F1H), a clinical‐grade, high‐throughput, hybridization capture‐based NGS assay for targeted sequencing of all exons of 406 genes, and RNA sequencing of 265 genes. F1H methods used have been previously reported in detail. Sequences were analyzed for genomic alterations including short variant alterations, copy number alterations, and gene rearrangements as described. 

Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) analysis was performed on a 4‐μm unstained section of representative formalin‐fixed, paraffin‐embedded tissue using a commercially available LSI break‐apart probe set (Vysis). The LSI SS18 Dual Color, Break Apart Rearrangement Probe consists of a mixture of two FISH DNA probes. The first probe, an ~650 kb probe labeled in SpectrumOrangeTM, extends distally from the SS18 gene. The second probe labeled in SpectrumGreenTM lies 3′ or proximal to the SS18 gene and is approximately 1040 kb in length. Hybridization signals were assessed in 200 interphase nuclei with strong, well‐delineated signals. Only nonoverlapping tumor nuclei with a complete set of signals were scored. The cut‐off level for scoring aberrations was 15% abnormal nuclei for the dual‐color SS18 FISH probe system. 

RESULTS 3.1 Case report The patient was a 60‐year‐old female who presented to an outside institution with a mass located on the volar aspect of the right hand. An excisional biopsy was performed to yield an ulcerative cutaneous malignant neoplasm composed of fascicles and nodular aggregates of malignant spindled and epithelioid cells within a hyalinized stroma dissecting through sclerotic collagen bundles, punctuated by confluent zones of coagulative tumor necrosis (Figure ). The tumor cells manifest nucleocytoplasmic pleomorphism and contained vesicular chromatin with irregular nuclear contours, pinpoint nucleoli and indistinct palely eosinophilic cytoplasm, and brisk mitotic rate (Figure ). By immunohistochemistry, the tumor cells were positive for keratins (AE1/E3 and Cam5.2), vimentin, p53, smooth muscle actin (SMA), TLE1 (multifocal, 40% overall), CMYC, BCL2 (focal), and negative for p40, p63, and S‐100. The INI1 stain was strongly and diffusely positive, retained in approximately 95% of neoplastic cells. Given the expression of smooth muscle actin in the tumor cells, we entertained the possibility of a cutaneous myoid/myogenic sarcoma with aberrant cytokeratin expression, but additional myogenic markers including smooth muscle heavy chain myosin (SMMS), H‐caldesmon, desmin, and myogenin were negative, significantly diminishing that diagnostic consideration. Furthermore, we considered the possibility of a cutaneous malignant vascular neoplasm, but again the vascular markers including ERG, CD31, and CD34, were all negative. The histomorphology appeared somewhat reminiscent of an epithelioid sarcoma and was initially labeled as malignant keratin‐positive spindled and epithelioid neoplasm, resembling epithelioid sarcoma. The neoplasm involved the surgical margins requiring multiple attempts for surgical re‐excision. A right forearm amputation was ultimately performed with axillary dissection. Examination of the surgical amputation revealed a 6.7 cm mass, recapitulating the histopathological features previously seen on excisional biopsy. All examined lymph nodes were negative for metastatic disease. One year following amputation of the primary lesion, surveillance positive emission tomography (PET) scan showed bilateral pulmonary nodules biopsied to reveal metastatic disease for which chemotherapy was initiated. One year following neoadjuvant therapy, metastatic lesions were identified within the brain by magnetic resonance imaging (MRI) requiring surgical resection and radiation therapy. Progression of pulmonary metastases was identified on computed tomography (CT) scan as well as newly identified metastatic lesions to the liver and kidney. Soft tissue subcutaneous nodules were also identified along the right and left anterior abdominal wall. Wide local excision of the subcutaneous nodules was performed to reveal three discrete, depressed, ulcerated lesions measuring 2.6 × 0.8 cm, 2.4 × 1.5 cm, and 4.6 × 4.1 cm. The histomorphology and immunophenotype were similar to the previous amputation specimen, confirming widely metastatic disease. The unusual histomorphology and inconclusive immunophenotypic profile, prompted NGS to further characterize the lesion. Results from the NGS panel identified an SS18::NEDD4 fusion gene with genomic coordinates 5′‐SS18 (ex1‐9 NM_005637)‐NEDD4 (ex14‐29 NM_006154) as well as mutations in the following genes: IDH1 R109K, AXIN1 A526V, CD36 splice sites 429+2T>C with the mutation allelic frequencies of 48.17%, 48.26%, and 47.90%, respectively (Figure ). Loss of CDKN2A/B was also identified. Rearrangement of SS18 was identified by FISH, further corroborating results of the NGS panel (Figure ). Taken together, the overall features were considered to be unclassified malignant cutaneous spindled and epithelioid sarcoma with a novel SS18::NEDD4 fusion gene. The aggressive clinical nature of this entity, however, was striking. Despite aggressive multimodality treatment approaches, the patient succumbed to her illness within 2 years of presentation. 

Case report The patient was a 60‐year‐old female who presented to an outside institution with a mass located on the volar aspect of the right hand. An excisional biopsy was performed to yield an ulcerative cutaneous malignant neoplasm composed of fascicles and nodular aggregates of malignant spindled and epithelioid cells within a hyalinized stroma dissecting through sclerotic collagen bundles, punctuated by confluent zones of coagulative tumor necrosis (Figure ). The tumor cells manifest nucleocytoplasmic pleomorphism and contained vesicular chromatin with irregular nuclear contours, pinpoint nucleoli and indistinct palely eosinophilic cytoplasm, and brisk mitotic rate (Figure ). By immunohistochemistry, the tumor cells were positive for keratins (AE1/E3 and Cam5.2), vimentin, p53, smooth muscle actin (SMA), TLE1 (multifocal, 40% overall), CMYC, BCL2 (focal), and negative for p40, p63, and S‐100. The INI1 stain was strongly and diffusely positive, retained in approximately 95% of neoplastic cells. Given the expression of smooth muscle actin in the tumor cells, we entertained the possibility of a cutaneous myoid/myogenic sarcoma with aberrant cytokeratin expression, but additional myogenic markers including smooth muscle heavy chain myosin (SMMS), H‐caldesmon, desmin, and myogenin were negative, significantly diminishing that diagnostic consideration. Furthermore, we considered the possibility of a cutaneous malignant vascular neoplasm, but again the vascular markers including ERG, CD31, and CD34, were all negative. The histomorphology appeared somewhat reminiscent of an epithelioid sarcoma and was initially labeled as malignant keratin‐positive spindled and epithelioid neoplasm, resembling epithelioid sarcoma. The neoplasm involved the surgical margins requiring multiple attempts for surgical re‐excision. A right forearm amputation was ultimately performed with axillary dissection. Examination of the surgical amputation revealed a 6.7 cm mass, recapitulating the histopathological features previously seen on excisional biopsy. All examined lymph nodes were negative for metastatic disease. One year following amputation of the primary lesion, surveillance positive emission tomography (PET) scan showed bilateral pulmonary nodules biopsied to reveal metastatic disease for which chemotherapy was initiated. One year following neoadjuvant therapy, metastatic lesions were identified within the brain by magnetic resonance imaging (MRI) requiring surgical resection and radiation therapy. Progression of pulmonary metastases was identified on computed tomography (CT) scan as well as newly identified metastatic lesions to the liver and kidney. Soft tissue subcutaneous nodules were also identified along the right and left anterior abdominal wall. Wide local excision of the subcutaneous nodules was performed to reveal three discrete, depressed, ulcerated lesions measuring 2.6 × 0.8 cm, 2.4 × 1.5 cm, and 4.6 × 4.1 cm. The histomorphology and immunophenotype were similar to the previous amputation specimen, confirming widely metastatic disease. The unusual histomorphology and inconclusive immunophenotypic profile, prompted NGS to further characterize the lesion. Results from the NGS panel identified an SS18::NEDD4 fusion gene with genomic coordinates 5′‐SS18 (ex1‐9 NM_005637)‐NEDD4 (ex14‐29 NM_006154) as well as mutations in the following genes: IDH1 R109K, AXIN1 A526V, CD36 splice sites 429+2T>C with the mutation allelic frequencies of 48.17%, 48.26%, and 47.90%, respectively (Figure ). Loss of CDKN2A/B was also identified. Rearrangement of SS18 was identified by FISH, further corroborating results of the NGS panel (Figure ). Taken together, the overall features were considered to be unclassified malignant cutaneous spindled and epithelioid sarcoma with a novel SS18::NEDD4 fusion gene. The aggressive clinical nature of this entity, however, was striking. Despite aggressive multimodality treatment approaches, the patient succumbed to her illness within 2 years of presentation. 

DISCUSSION Our case is the first report of a non‐SS malignant neoplasm identified in the literature to harbor the novel SS18::NEDD4 fusion gene. Argani et al. recently described the first primary renal SS with positive immunoreactivity for TLE1. Also, unique to this case was the bland histomorphology defined by a predominantly hypocellular spindle‐cell proliferation within a myxoid stroma, akin to the myxoid variant of myxoid SS. The genomic coordinates of the index case corresponding to the breakpoints in exons 1‐9 of SS18 and exons 14‐29 of NEDD4 produce an identical SS18::NEDD4 fusion to that identified in the primary renal SS. Although the mechanisms by which the novel SS18::NEDD4 gene fusion have not been fully elucidated, individual perturbations in SS18‐ and NEDD4‐mediated signaling pathways are recognized for promoting human cancer and tumorigenesis. The SS18 gene is ubiquitously expressed in normal human tissue and encodes for the SS18 subunit of mammalian SWI/SNF (BAF) chromatin remodeling complexes which functions to maintain balance in both gene activation and repression. , , The SS18::SSX fusion gene results from the replacement of the last eight C‐terminal amino acids of SS18 by 78 C‐terminal amino acids of SSX. The fusion product produces the SS18::SSX oncogene which retains the ability of SS18 to incorporate into BAF complexes, however, displacing the BAF47 (SMARCB1/SNF5/INI1) tumor suppressor subunit. Neural precursor cell‐expressed developmentally downregulated gene 4 ( NEDD4 ) is a member of the E3 ubiquitin ligases. The NEDD4 subfamily proteins are key regulators of cell growth, endocytosis, and modulation of neuromuscular junctions. , Recent studies have emerged to reveal the potential role of NEDD4 in human cancer by modulating ubiquitination‐dependent degradation of downstream substrates involved in carcinogenesis and tumorigenesis including IGF‐IR, FGFR1, EGFR, VEGFR2, HER3/Erb3, PTEN, Notch, phosphor‐Akt, c‐Myc, Ras, and mdm2. , Increased expressions of NEDD4 has been identified by immunohistochemical staining in gastric and colorectal cancer, hepatocellular carcinoma, non‐small‐cell lung carcinoma, invasive ductal carcinoma, and endometrial adenocarcinoma. , , However, NEDD4 rearrangements have not yet been identified in the aforementioned entities. The spectrum of cutaneous spindle cell malignancies spans the entire gamut from sarcomatoid carcinoma and spindle cell/desmoplastic melanomas to frank sarcomas, with differing prognostic and therapeutic implications. From a purely molecular perspective, we had considered the possibility of SS, but the morphologic phenotype bears no resemblance to SS. SS is an aggressive malignant mesenchymal neoplasm of uncertain origin, characterized by reciprocal t(X,18)(p11;q11) resulting in SS18::SSX1/2 or 4 in the vast majority of cases. It can present as a monomorphic proliferation of spindle cells or as a biphasic neoplasm with epithelioid differentiation. There are three principal types including: monophasic fibrous type, biphasic, and poorly differentiated type. Histologically, they are composed of sweeping fascicles of spindle cells with prominent nuclear hyperchromasia. Variable collagen deposition can be seen in the monophasic type. Biphasic SS may contain glandular components, and poorly differentiated round cell morphology can be seen in the poorly differentiated type. The immunohistochemical profile demonstrates diffuse positivity for SS18‐SSX and variable reactivity for keratins and/or epithelial membrane antigen. TLE1 has been regarded as a highly sensitive and relatively specific marker of synovia sarcomas with strong and diffuse positive immunoreactivity. However, the heterogeneity and low specificity of TLE1 expression in non‐SSs should be recognized in the differential diagnosis including epithelioid sarcoma which has shown multifocal positivity in 2 out of 6 cases with 2+ and 3+ staining in a large study of 163 soft tissue tumors. In the index case, the leading differential diagnosis based on histomorphology alone was epithelioid sarcoma. Epithelioid sarcoma is a relatively rare malignant mesenchymal neoplasm defined by epithelioid cytomorphology and evidence of epithelial differentiation measured by positive keratin expression by immunohistochemistry and loss of INI1 (SMARCB1). The classic subtype accounts for two‐thirds of epithelioid sarcoma and most commonly occurs in adolescents and young adults arising within the distal extremities, in particular, the hand and fingers. The proximal variant of epithelioid sarcoma exhibits a pleomorphic and anaplastic morphology with aggressive biology and commonly arises within the deep soft tissues of the pelvis, inguinal region, axilla, and flank. Rarely, the spindled histomorphology may dominate and has been reported as fibroma‐like epithelioid sarcoma. Recurrence of epithelioid sarcoma is common and may occur in up to 70% of cases. Metastasis of epithelioid sarcoma, including distant metastasis to lymph nodes, visceral organs, and skin, can occur in up to 50% of cases and is uniformly fatal. Given the intimate association of the neoplastic cells with the overlying skin, we considered the possibility of sarcomatoid squamous cell carcinoma. Sarcomatoid squamous cell carcinoma shares overlapping features with sarcomas. However, the growth pattern, lack of background keratinocyte atypia from chronic ultraviolet light exposure, and lack of expression markers such as p40, p63, CK5/6 makes this an unlikely consideration. Furthermore, the genomic profile, lacking chronic ultraviolent light molecular signature, low tumor mutational burden, and the clinical behavior of widespread cutaneous and visceral metastases (and sparing lymph nodes) suggests that the index case was mesenchymal in origin rather than epithelial in nature. Interestingly, our case demonstrated multiple additional genetic aberrations, specifically, single nucleotide variants, SNV, involving AXIN1 A526V, CD36 splice site 429+2T>C, and IDH1 R109K; all of which have been identified in the literature. However, the pathological significance of the SNVs in the index case is unknown. Loss of CDKN2A/B has been identified in soft tissue sarcomas, B‐cell non‐Hodgkin lymphoma, and astrocytoma. Unsupervised clustering of transcriptome profiles by RNA‐sequencing in the previously reported case of SS18::NEDD4 fusion‐positive primary renal SS revealed close grouping with 3 SSs within the database providing further evidence to support the diagnosis. While clustering analysis was not performed for the index case, we note that the genomic breakpoints for the SS18 and NEDD4 genes were different in both cases which may result in different domain compositions of the fusion oncoprotein. Obviously, whether they cluster together or not remains speculative. Importantly, the resultant morphologic and immunophenotypic profile and biologic behavior in our index differs greatly from the previously reported case of SS. This case underscores the significance of molecular pleiotropism and the inherent promiscuity of similar fusion genes that arise in tumors with divergent histotypes and biological behaviors, with increasing clinical use of NGS platform. It is also conceivable that additional factors including epigenetic and stochastic genomic events may play a role. The detection of a CDKN2A deletion has been recognized in a recent study as a potential biomarker for poor prognosis in soft tissue sarcomas, including subtypes such as SS, malignant peripheral nerve sheath tumor, sarcoma NOS, and undifferentiated pleomorphic sarcoma. Finally, we considered the possibility of spindle cell malignant melanoma with aberrant cytokeratin reactivity. The marked morphologic and immunophenotypic diversity in malignant melanoma is well documented. Indeed, malignant melanomas have been known to express a wide variety of markers including cytokeratin, desmin, smooth muscle actin, amongst others. However, in the majority of cases, the melanocytic immunophenotypic profile is retained and lost after dedifferentiation to a sarcomatoid phenotype. In our case, the uniform cytomorphology (lacking a dedifferentiated component) and absence of melanocytic expression greatly diminished this diagnostic consideration. In summary, we report a case of an unclassified keratin positive cutaneous spindled and epithelioid sarcoma, resembling epithelioid sarcoma, and harboring the SS18‐NEDD4 fusion with a very aggressive clinical behavior in a 60‐year‐old female. Our case displayed an aggressive and fatal clinical course different from that of the previously reported primary renal SS harboring an identical SS18‐NEDD4 fusion gene. The mechanisms are not yet clear. However, it is conceivable that the constellation of multiple genetic hits including CDKN2A mutation may have played a role in the aggressive biology. Future studies are warranted to further characterize this novel entity. 

Obiajulu Hans Iwenofu , conceived the project. Ashley Patton , Steve Oghumu , and Obiajulu Hans Iwenofu provided essential tools/data. AP & OHI wrote the paper. All authors approved the final manuscript. 

The authors declare no conflicts of interest. 

