Comprehensive genomic profiling of EWSR1/FUS::CREB translocation-associated tumors uncovers prognostically significant recurrent genetic alterations and methylation-transcriptional correlates.

To elucidate the mechanisms underlying the divergent clinicopathologic spectrum of EWSR1/FUS::CREB translocation-associated tumors, we performed a comprehensive genomic analysis of fusion transcript variants, recurrent genetic alterations (mutations, copy number alterations), gene expression, and methylation profiles across a large cohort of tumor types. The distribution of the EWSR1/FUS fusion partners-ATF1, CREB1, and CREM-and exon involvement was significantly different across different tumor types. Our targeted sequencing showed that secondary genetic events are associated with tumor type rather than fusion type. Of the 39 cases that underwent targeted NGS testing, 18 (46%) had secondary OncoKB mutations or copy number alterations (29 secondary genetic events in total), of which 15 (52%) were recurrent. Secondary recurrent, but mutually exclusive, TERT promoter and CDKN2A mutations were identified only in clear cell sarcoma (CCS) and associated with worse overall survival. CDKN2A/B homozygous deletions were recurrent in angiomatoid fibrous histiocytoma (AFH) and restricted to metastatic cases. mRNA upregulation of MITF, CDH19, PARVB, and PFKP was found in CCS, compared to AFH, and correlated with a hypomethylated profile. In contrast, S100A4 and XAF1 were differentially upregulated and hypomethylated in AFH but not CCS. Unsupervised clustering of methylation profiles revealed that CREB family translocation-associated tumors form neighboring but tight, distinct clusters. A sarcoma methylation classifier was able to accurately match 100% of CCS cases to the correct methylation class; however, it was suboptimal when applied to other histologies. In conclusion, our comprehensive genomic profiling of EWSR1/FUS::CREB translocation-associated tumors uncovered mostly histotype, rather than fusion-type associated correlations in transcript variants, prognostically significant secondary genetic alterations, and gene expression and methylation patterns.

Generation of human embryonic stem cell models to exploit the EWSR1-CREB fusion promiscuity as a common pathway of transformation in human tumors.

Chromosomal translocations constitute driver mutations in solid tumors and leukemias. The mechanisms of how related or even identical gene fusions drive the pathogenesis of various tumor types remain elusive. One remarkable example is the presence of EWSR1 fusions with CREB1 and ATF1, members of the CREB family of transcription factors, in a variety of sarcomas, carcinomas and mesotheliomas. To address this, we have developed in vitro models of oncogenic fusions, in particular, EWSR1-CREB1 and EWSR1-ATF1, in human embryonic stem (hES) cells, which are capable of multipotent differentiation, using CRISPR-Cas9 technology and HDR together with conditional fusion gene expression that allows investigation into the early steps of cellular transformation. We show that expression of EWSR1-CREB1/ATF1 fusion in hES cells recapitulates the core gene signatures, respectively, of angiomatoid fibrous histiocytoma (AFH) and gastrointestinal clear cell sarcoma (GI-CCS), although both fusions lead to cell lethality. Conversely, expression of the fusions in hES cells differentiated to mesenchymal progenitors is compatible with prolonged viability while maintaining the core gene signatures. Moreover, in the context of a mesenchymal lineage, the proliferation of cells expressing the EWSR1-CREB1 fusion is further extended by deletion of the tumor suppressor TP53. We expect the generation of isogenic lines carrying oncogenic fusions in various cell lineages to expand our general understanding of how those single genetic events drive tumorigenesis while providing valuable resources for drug discovery.

Unclassified low grade spindle cell sarcoma with storiform pattern characterized by recurrent novel EWSR1/FUS-NACC1 fusions.

Despite extraordinary advances in the molecular characterization of soft tissue tumors as a result of the widespread application of next generation sequencing in clinical practice, a subset of lesions remain difficult to diagnose. In this study we describe 3 unclassified spindle cell sarcomas with a monomorphic cytomorphology and distinctive storiform growth, characterized by novel fusions between EWSR1 or FUS1, and NACC1 genes. The tumors occurred in 3 young adult females (age range: 29-31) involving deep soft tissues, two located in the lower extremity and one in the abdominal wall. All three tumors showed patchy positivity for S100 protein, while being negative for SOX10 and retained H3K27me3 expression. All cases were negative for epithelial or muscle markers. As the findings were non-specific, molecular studies using targeted panels of RNA sequencing were performed, including one case tested by TruSight RNA Fusion Panel and 2 cases by Archer FusionPlex. The results showed 2 cases were positive for FUS-NACC1 and one for EWSR1-NACC1 fusions. These findings were further confirmed by FISH using custom BAC probes for a dual-color fusion assay. These results suggest the possibility of a previously undescribed soft tissue neoplasm characterized by a uniform spindle cell phenotype arranged in a storiform and fascicular pattern, expressing S100 protein and harboring NACC1-related fusions. The biologic behavior of this tumor remains to be determined.

Hyalinizing epithelioid tumors with OGT-FOXO fusions. A case report of a non-acral soft tissue mass harboring a novel FOXO4 gene rearrangement.

Recurrent fusions between OGT and members of the Forkhead box (FOXO) family of genes have been recently described in three cases of hyalinizing epithelioid acral soft tissue tumors in young adults showing co-expression for EMA and CD34. Despite the lack of an established myoepithelial lineage by immunohistochemistry, these lesions have been labeled as myoepithelioma-like due to their epithelioid phenotype and sclerotic background. In this study, we report a novel FOXO4-OGT fusion identified by targeted RNA sequencing in an unclassified shoulder soft tissue mass in a 40-year-old male. The tumor showed nodular foci of increased cellularity in a uniformly hyalinized background. The neoplastic cells were mainly epithelioid and focally spindled, with eosinophilic cytoplasm and indented nuclei with mild atypia. The tumor lacked significant mitotic activity and necrosis. Immunohistochemically, the tumor showed variable positivity for EMA, pan-CK, CD34, ERG and FLI1, while it was negative for CD31, S100, SOX10, desmin, and MUC4. INI1 expression was retained. Due to its unusual histology and conflicting immunoprofile, TruSight RNA fusion panel sequencing was performed which revealed a fusion between FOXO4 exon 2 to OGT exon 2. This is the first example of a soft tissue lesion harboring OGT-related fusions occurring in a non-acral location and associated with FOXO4 gene. Its line of differentiation and biologic potential remain uncertain.

Pediatric Mesothelioma With ALK Fusions: A Molecular and Pathologic Study of 5 Cases.

Pediatric mesotheliomas are rare and their pathogenesis remains undefined. In this study, we report 5 cases of malignant mesothelioma in children, characterized by fusions involving the anaplastic lymphoma kinase (ALK) gene. Four cases occurred in females involving the abdominal cavity and were characterized by a pure epithelioid morphology. The fifth arose in the tunica vaginalis of a 15-year-old male and displayed a biphasic epithelioid-sarcomatoid phenotype. All cases demonstrated the classic morphologic and immunohistochemical features of malignant mesothelioma, including tubulopapillary architecture and cuboidal epithelioid cells with eosinophilic cytoplasm and uniform nuclei with vesicular chromatin. Immunohistochemically, all cases showed labeling for ALK, cytokeratins, WT1, and calretinin, while lacking expression of adenocarcinoma immunomarkers. Four cases demonstrated weak-moderate labeling for PAX8 protein, which resulted in diagnostic challenges with primary peritoneal serous carcinoma. The ALK genetic abnormalities were investigated by a combination of molecular methods. Archer FusionPlex was performed in 2 cases, showing fusions between ALK with either STRN or TPM1 genes, resulting in a transcript that retained the ALK kinase domain. One case was further studied by DNA targeted sequencing, but no additional genetic alterations were observed. In 1 case, cytogenetic analysis showed the presence of a t(2;15)(p23;q22) and fluorescence in situ hybridization confirmed the ALK gene break-apart. In the remaining 2 cases, ALK gene rearrangements were demonstrated by fluorescence in situ hybridization. Unlike adult mesotheliomas, which are tightly linked to asbestos exposure, often show loss of BAP1 expression and have complex karyotypes, ALK-rearranged mesothelioma appears to be similar to other fusion-positive mesotheliomas, such as those harboring EWSR1/FUS-ATF1 fusions, sharing significant morphologic overlap, occurring in young patients and displaying a simple, translocation-driven genetic profile.

Novel GATA6-FOXO1 fusions in a subset of epithelioid hemangioma.

The genetic hallmark of epithelioid hemangioma (EH) is the presence of recurrent gene fusions involving FOS and FOSB transcription factors, which occur in one-third of the cases. Certain clinical, pathologic, and genotypic correlations have been described, with FOS-related fusions being more often detected in skeletal and cellular variants of EH, while FOSB gene rearrangements are more commonly associated with atypical histologic features and penile location. These fusions are infrequently detected in the cutaneous or head and neck EH. Overall, two-thirds of EH lack these canonical fusions and remain difficult to classify, especially when associated with atypical features and/or clinical presentations. Triggered by an index case of an intravascular soft tissue EH with a novel GATA6-FOXO1 gene fusion by targeted RNA sequencing (Archer® FusionPlex® Sarcoma Panel), we have investigated 27 additional EH cases negative for FOS and FOSB gene rearrangements for this novel abnormality to determine its recurrent potential, and its association with clinical and pathologic features. Four additional EH cases were found to display GATA6-FOXO1 fusions (18%). There were three females and two males, with a mean age of 32 years old. Three lesions occurred in the head and neck (dura, nasopharyngeal, and cheek), one in the back and one in the leg. Two of these lesions were cutaneous and one was intravascular in the subcutis of the leg. Microscopically, the tumors showed a variegated morphology, with alternating vasoformative and solid components, extravasated red blood cells and mild to moderate cytologic atypia. None showed brisk mitotic activity or necrosis. Tumors were negative for FOS and FOSB by immunohistochemistry. In conclusion, we report a new GATA6-FOXO1 fusion in a subset of EH, with a predilection for skin, and head and neck location. The relationship of this novel molecular subset with the more common FOS/FOSB fusion-positive EH remains to be determined.

Epithelioid hemangioma of bone harboring FOS and FOSB gene rearrangements: A clinicopathologic and molecular study.

The diagnosis of epithelioid hemangioma (EH) remains challenging due to its rarity, worrisome histologic features, and locally aggressive clinical and radiographic presentation. Especially in the bone, EH can be misdiagnosed as a malignant vascular neoplasm due its lytic, often destructive or multifocal growth, as well as atypical morphology. The discovery of recurrent FOS and FOSB gene fusions in the pathogenesis of most EH has strengthened its stand-alone classification, distinct from other malignant epithelioid vascular lesions, such as epithelioid hemangioendothelioma or angiosarcoma. In this study we investigate a group of molecularly confirmed skeletal EH by the presence of FOS or FOSB gene rearrangements to better define its clinical and pathologic characteristics within a homogenous molecular subset. The cohort included 38 patients (25 males, 13 females), with a mean age at diagnosis of 38 years (range, 4-75). Regional, multifocal presentation was noted in 10 cases. Only six cases were correctly recognized as EH by the referring institutions, while most were misdiagnosed as other vascular tumors. Of the 17 patients with follow-up data available, five patients (29%) developed local recurrence after marginal en bloc excision (n = 3) or curettage (n = 2). Local recurrence-free survival rates were 84% at 3 years and 38% at 5 years. No metastasis or disease-related death was identified. Imaging studies exhibited no specific features, showing cortical bone destruction and soft-tissue extension in 14 (38%) cases. FOS gene rearrangements were detected in 28 (74%) of cases, while FOSB rearrangements in 10 (26%) cases. Our results highlight the significant challenges encountered in establishing a correct diagnosis exclusive of the molecular testing, mainly due to its overlap to other malignant epithelioid vascular tumors. Skeletal EH emerges as a genetically defined locally aggressive vascular neoplasm, with a high rate of local recurrence, but lacking the propensity for distant spread.

Recurrent YAP1 and MAML2 Gene Rearrangements in Retiform and Composite Hemangioendothelioma.

Retiform and composite hemangioendotheliomas (CHEs) are both locally aggressive, rarely metastasizing vascular neoplasms characterized by arborizing vascular channels lined by endothelial cells with a hobnail morphology. CHE displays additional cytologic and architectural components, including often vacuolated epithelioid cells, solid areas, or features reminiscent of well-differentiated angiosarcoma. Triggered by an index case of a soft tissue retiform hemangioendothelioma (RHE) which revealed a YAP1-MAML2 gene fusion by targeted RNA sequencing, we sought to investigate additional cases in this morphologic spectrum for this genetic abnormality. A total of 24 cases, 13 RHE and 11 CHE involving skin and soft tissue were tested by fluorescence in situ hybridization using custom BAC probes for rearrangements involving these genes. An additional visceral CHE with neuroendocrine differentiation was tested by targeted RNA sequencing. Among the soft tissue cohort, 5/13 (38%) RHE and 3/11 (27%) CHE showed YAP1 gene rearrangements, with 5 cases showing a YAP1-MAML2 fusion, including all 3 CHE. The single neuroendocrine CHE showed the presence of a PTBP1-MAML2 fusion. All YAP1-positive CHE lesions occurred in female children at acral sites, compared with fusion-negative cases which occurred in adults, with a wide anatomic distribution. YAP1-positive RHE occurred preferentially in males and lower limb, compared with negative cases. These results suggest that RHE and CHE represent a morphologic continuum, sharing abnormalities in YAP1 and MAML2 genes. In contrast, the neuroendocrine CHE occurring in a 37-year-old male harbored a distinct PTBP1-MAML2 fusion and showed aggressive clinical behavior (pancreatic mass with multiple liver and lung metastases). These preliminary findings raise the possibility that neuroendocrine CHE may be genetically distinct from the conventional RHE/CHE spectrum. Further studies are needed to investigate the pathogenetic relationship of fusion-negative cases with this subset and, less likely, with other members of the HE family of tumors.

EWSR1/FUS-CREB fusions define a distinctive malignant epithelioid neoplasm with predilection for mesothelial-lined cavities.

Gene fusions constitute pivotal driver mutations often encoding aberrant chimeric transcription factors. However, an increasing number of gene fusion events have been shown not to be histotype specific and shared among different tumor types, otherwise completely unrelated clinically or phenotypically. One such remarkable example of chromosomal translocation promiscuity is represented by fusions between EWSR1 or FUS with genes encoding for CREB-transcription factors family (ATF1, CREB1, and CREM), driving the pathogenesis of various tumor types spanning mesenchymal, neuroectodermal, and epithelial lineages. In this study, we investigate a group of 13 previously unclassified malignant epithelioid neoplasms, frequently showing an epithelial immunophenotype and marked predilection for the peritoneal cavity, defined by EWSR1/FUS-CREB fusions. There were seven females and six males, with a mean age of 36 (range 9-63). All except three cases occurred intra-abdominally, including one each involving the pleural cavity, upper, and lower limb soft tissue. All tumors showed a predominantly epithelioid morphology associated with cystic or microcystic changes and variable lymphoid cuffing either intermixed or at the periphery. All except one case expressed EMA and/or CK, five were positive for WT1, while being negative for melanocytic and other mesothelioma markers. Nine cases were confirmed by various RNA-sequencing platforms, while in the remaining four cases the gene rearrangements were detected by FISH. Eleven cases showed the presence of CREM-related fusions (EWSR1-CREM, 7; FUS-CREM, 4), while the remaining two harbored EWSR1-ATF1 fusion. Clinically, seven patients presented with and/or developed metastases, confirming a malignant biologic potential. Our findings expand the spectrum of tumors associated with CREB-related fusions, defining a novel malignant epithelioid neoplasm with an immunophenotype suggesting epithelial differentiation. This entity appears to display hybrid features between angiomatoid fibrous histiocytoma (cystic growth and lymphoid cuffing) and mesothelioma (peritoneal/pleural involvement, epithelioid phenotype, and cytokeratin and WT1 co-expression).

Undifferentiated round cell sarcomas with novel SS18-POU5F1 fusions.

Despite significant recent advances in characterizing the molecular pathogenesis of undifferentiated round cell neoplasms, rare cases remain unclassified. Here, we report two distinctive undifferentiated round cell tumors occurring in young adults. One tumor presented intrabdominally and the other arose within the abdominal wall. One patient died of disease following local and distance recurrence, despite aggressive chemotherapy and radiotherapy. Morphologically, both tumors were similarly composed of primitive round to epithelioid cells arranged in nests, sheets, and trabecular patterns. The cytoplasm was scant and amphophilic, while the nuclei were round and uniform with brisk mitotic activity. Focal necrosis was present. Immunohistochemically, both tumors were variably positive for S100 and EMA, and one case focally expressed cytokeratin and TLE1. Targeted RNA sequencing revealed in both an identical SS18-POU5F1 fusion gene. Fluorescence in situ hybridization was performed which confirmed SS18 and POU5F1 gene rearrangements. Expression data, relative to over 200 other mesenchymal neoplasms that had undergone targeted RNA sequencing on the same platform, suggested the SS18-POU5F1 tumors cluster with EWSR1/FUS-POU5F1-positive myoepithelial tumors. In view of our limited sample size, additional studies are needed to characterize the breadth of clinical and pathologic findings in these neoplasms. In addition, further investigation is necessary to determine whether this entity represents a clinically aggressive and phenotypically undifferentiated variant of myoepithelial tumors, or perhaps an altogether novel category of undifferentiated round cell sarcoma.

Soft tissue tumors characterized by a wide spectrum of kinase fusions share a lipofibromatosis-like neural tumor pattern.

Gene fusions resulting in oncogenic activation of various receptor tyrosine kinases, including NTRK1-3, ALK, and RET, have been increasingly recognized in soft tissue tumors (STTs), displaying a wide morphologic spectrum and therefore diagnostically challenging. A subset of STT with NTRK1 rearrangements were recently defined as lipofibromatosis-like neural tumors (LPFNTs), being characterized by mildly atypical spindle cells with a highly infiltrative growth in the subcutis and expression of S100 and CD34 immunostains. Other emerging morphologic phenotypes associated with kinase fusions include infantile/adult fibrosarcoma and malignant peripheral nerve sheath tumor-like patterns. In this study, a large cohort of 73 STT positive for various kinase fusions, including 44 previously published cases, was investigated for the presence of an LPFNT phenotype, to better define the incidence of this distinctive morphologic pattern and its relationship with various gene fusions. Surprisingly, half (36/73) of STT with kinase fusions showed at least a focal LPFNT component defined as >10%. Most of the tumors occurred in the subcutaneous tissues of the extremities (n = 25) and trunk (n = 9) of children or young adults (<30 years old) of both genders. Two-thirds (24/36) of these cases showed hybrid morphologies with alternating LPFNT and solid areas of monomorphic spindle to ovoid tumor cells with fascicular or haphazard arrangement, while one-third (12/36) had pure LPFNT morphology. Other common histologic findings included lymphocytic infiltrates, staghorn-like vessels, and perivascular or stromal hyalinization, especially in hybrid cases. Mitotic activity was generally low (<4/10 high power fields in 81% cases), being increased only in a minority of cases. Immunoreactivity for CD34 (92% in hybrid cases, 89% in pure cases) and S100 (89% in hybrid cases, 64% in pure cases) were commonly present. The gene rearrangements most commonly involved NTRK1 (75%), followed by RET (8%) and less commonly NTRK2, NTRK3, ROS1, ALK, and MET.

NTRK3 overexpression in undifferentiated sarcomas with YWHAE and BCOR genetic alterations.

The BCOR family of tumors includes a number of undifferentiated sarcomas, occurring in various age groups and anatomic sites, characterized by a spindle and round cell phenotype and diffuse immunoreactivity for BCOR. Prior RNA sequencing data revealed that NTRK3 was a top-upregulated gene in BCOR-CCNB3 sarcomas. In this study, we investigate a large cohort of tumors harboring BCOR/YWHAE genetic alterations for NTRK3 upregulation at both the mRNA and protein levels, compared with other sarcoma types. Pan-Trk immunohistochemistry was assessed for intensity and extent. A correlation between NTRK3 expression and the type of BCOR alteration and BCOR immunoreactivity was also performed. Most soft tissue undifferentiated round cell sarcomas with YWHAE or BCOR rearrangements or BCOR internal tandem duplications (ITD) showed NTRK3, but not NTRK1 or NTRK2, upregulation by RNA sequencing data analysis. Cytoplasmic pan-Trk immunoreactivity was also observed in most soft tissue round cell sarcomas with YWHAE rearrangements (100%), BCOR ITD (80%), and BCOR-CCNB3 fusions (67%), as well as clear cell sarcomas of kidney (75%), another BCOR family tumor, and ossifying fibromyxoid tumors with ZC3H7B-BCOR fusion (100%), with variable staining intensity and extent. Pan-Trk staining was also seen in solitary fibrous tumors (100%) and less frequently in synovial sarcoma and Ewing sarcoma, but rarely in other sarcomas tested. Tumors harboring rare fusion variants of BCOR, such as BCOR-CHD9, a novel fusion identified by targeted RNA sequencing, and KMT2D-BCOR, were also positive for pan-Trk staining and NTRK3 overexpression. In conclusion, NTRK3 upregulation resulting in pan-Trk overexpression is common in the BCOR family of tumors as well as in subsets of BCOR-expressing sarcomas through alternative mechanisms. The therapeutic implication of this finding awaits further investigation.

A morphologic and molecular reappraisal of myoepithelial tumors of soft tissue, bone, and viscera with EWSR1 and FUS gene rearrangements.

Myoepithelial tumors (MET) represent a clinicopathologically heterogeneous group of tumors, ranging from benign to highly aggressive lesions. Although MET arising in soft tissue, bone, or viscera share morphologic and immunophenotypic overlap with their salivary gland and cutaneous counterparts, there is still controversy regarding their genetic relationship. Half of MET of soft tissue and bone harbor EWSR1 or FUS related fusions, while MET arising in the salivary gland and skin often show PLAG1 and HMGA2 gene rearrangements. Regardless of the site of origin, the gold standard in diagnosing a MET relies on demonstrating its "myoepithelial immunophenotype" of positivity for EMA/CK and S100 protein or GFAP. However, the morphologic spectrum of MET in soft tissue and bone is quite broad and the above immunoprofile is nonspecific, being shared by other pathogenetically unrelated neoplasms. Moreover, rare MET lack a diagnostic immunoprofile but shows instead the characteristic gene fusions. In this study, we analyzed a large cohort of 66 MET with EWSR1 and FUS gene rearrangements spanning various clinical presentations, to better define their morphologic spectrum and establish relevant pathologic-molecular correlations. Genetic analysis was carried out by FISH for EWSR1/FUS rearrangements and potential partners, and/or by targeted RNA sequencing. Then, 82% showed EWSR1 rearrangement, while 18% had FUS abnormalities. EWSR1-POU5F1 occurred with predilection in malignant MET in children and young adults and these tumors had nested epithelioid morphology and clear cytoplasm. In contrast, EWSR1/FUS-PBX1/3 fusions were associated with benign and sclerotic spindle cell morphology. Tumors with EWSR1-KLF17 showed chordoma-like morphology. Our results demonstrate striking morphologic-molecular correlations in MET of bone, soft tissue and viscera, which might have implications in their clinical behavior.

A novel RBMX-TFE3 gene fusion in a highly aggressive pediatric renal perivascular epithelioid cell tumor.

We report an Xp11 translocation perivascular epithelioid cell tumor (PEComa) with a novel RBMX-TFE3 gene fusion, resulting from a paracentric X chromosome inversion, inv(X)(p11;q26). The neoplasm occurred in an otherwise healthy 12-year-old boy who presented with a large left renal mass with extension into the inferior vena cava. The patient was found to have multiple pulmonary metastases at diagnosis and died of disease 3 months later. The morphology (epithelioid clear cells with alveolar and nested architecture) and immunophenotype (TFE3 and HMB45 strongly positive; actin, desmin, and PAX8 negative) was typical of an Xp11 translocation PEComa; however, TFE3 rearrangement was initially not detected by routine TFE3 break-apart fluorescence in situ hybridization (FISH). Further RNA sequencing revealed a novel RBMX-TFE3 gene fusion, which was subsequently confirmed by fusion assay FISH, using custom design RBMX and TFE3 come-together probes. This report describes a novel TFE3 gene fusion partner, RBMX, in a pediatric renal PEComa patient associated with a fulminant clinical course. As documented in other intrachromosomal Xp11.2 inversions, such as fusions with NONO, RBM10, or GRIPAP1 genes, the TFE3 break-apart might be below the FISH resolution, resulting in a false negative result.

Genetic diversity in alveolar soft part sarcoma: A subset contain variant fusion genes, highlighting broader molecular kinship with other MiT family tumors.

Alveolar soft part sarcoma (ASPS) is a rare malignancy that, since its initial description, remains a neoplasm of uncertain histogenesis. The disease-defining molecular event characterizing the diagnosis of ASPS is the ASPSCR1-TFE3 fusion gene. Following identification of an index case of ASPS with a novel TFE3 fusion partner, we performed a retrospective review to determine whether this represents an isolated event. We identified two additional cases, for a total of three cases lacking ASPSCR1 partners. The average patient age was 46 years (range, 17-65); two patients were female. The sites of origin included the transverse colon, foot, and dura. Each case exhibited a histomorphology typical of ASPS, and immunohistochemistry was positive for TFE3 in all cases. Routine molecular testing of the index patient demonstrated a HNRNPH3-TFE3 gene fusion; the remaining cases were found to have DVL2-TFE3 or PRCC-TFE3 fusion products. The latter two fusions have previously been identified in renal cell carcinoma; to our knowledge, this is the first report of a HNRNPH3-TFE3 gene fusion. These findings highlight a heretofore underrecognized genetic diversity in ASPS, which appears to more broadly molecularly overlap with that of translocation-associated renal cell carcinoma and PEComa. These results have immediate implications in the diagnosis of ASPS since assays reliant upon ASPSCR1 may yield a false negative result. While these findings further understanding of the molecular pathogenesis of ASPS, issues related to the histogenesis of this unusual neoplasm remain unresolved.

Clinical and molecular characterization of primary sclerosing epithelioid fibrosarcoma of bone and review of the literature.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare sarcoma subtype characterized by monomorphic epithelioid cells embedded in a densely sclerotic collagenous matrix. The overwhelming majority of tumors arise in soft tissues; however, rare cases have been documented to occur primarily in bone. The hallmarks of soft tissue SEF include MUC4 immunoreactivity and the presence of an EWSR1-CREB3L1 fusion. Rare cases with alternative fusions have also been reported such as EWSR1-CREB3L2 and FUS-CREB3L2 transcripts. The molecular alterations of skeletal SEF have not been well-defined, with only rare cases analyzed to date. In this study we investigated the clinicopathologic and molecular features of seven patients presenting with primary osseous SEF. There were 3 males and 4 females, with a mean age at diagnosis of 38 years. All cases had microscopic features within the histologic spectrum of SEF and showed strong and diffuse MUC4 positivity, while lacking SATB2 expression. However, due to its unusual presentation within bone, four cases were initially misinterpreted as either osteosarcoma, Ewing sarcoma or chondroblastoma. Half of the patients with follow-up data developed metastasis. The cases were tested by targeted RNA sequencing, MSK-IMPACT, and/or fluorescence in situ hybridization, showing EWSR1-CREB3L1 in six cases and EWSR1-CREB3L2 in one case. The fusion transcripts were composed of EWSR1 exon 11 to either exon 6 of CREB3L1 or CREB3L2. In summary, due to their rarity in the bone, skeletal SEF are often misdiagnosed, resulting in inadequate treatment modalities. Similar to their soft tissue counterpart, bone SEF follow an aggressive clinical behavior and show similar EWSR1-CREB3L1/CREB3L2 fusions.

A molecular study of synovial chondromatosis.

Synovial chondromatosis (SC) is a rare benign cartilaginous neoplasm in which recurrent fibronectin 1 (FN1) and activin receptor 2A (ACVR2A) gene rearrangements have been recently reported. Triggered by a case of malignant transformation in SC (synovial chondrosarcoma) showing a novel KMT2A-BCOR gene fusion by targeted RNA sequencing, we sought to evaluate the molecular abnormalities in a cohort of 27 SC cases using a combined methodology of fluorescence in situ hybridization (FISH) and/or targeted RNA sequencing. Results showed that FN1 and /or ACVR2A gene rearrangements were noted in 18 cases (67%), with an FN1-ACVR2A fusion being confirmed in 15 (56%) cases. Two cases showed only FN1 gene rearrangement, without other abnormalities. A novel FN1-NFATc2 gene fusion was noted in one case by RNA sequencing. The remaining nine cases showed no abnormalities in FN1 and ACVR2A genes. No additional cases showed BCOR gene alterations. In conclusion, this study confirms that FN1-ACVR2A fusion is the leading pathogenetic event in SC, at even higher frequency than previously reported. FISH methodology emerges as an appropriate tool in the identification of FN1 and ACVR2A gene abnormalities, which can be used in challenging cases. Further studies are needed to determine the recurrent potential of BCOR abnormalities in this disease.

Novel SRF-ICA1L Fusions in Cellular Myoid Neoplasms With Potential For Malignant Behavior.

Pericytic tumors comprise a histologic continuum of neoplasms with perivascular myoid differentiation, which includes glomus tumors, myopericytoma, myofibroma, and angioleiomyoma. Despite their morphologic overlap, recent data suggest a dichotomy in their genetic signatures, including recurrent NOTCH gene fusions in glomus tumors and PDGFRB mutations in myofibromas and myopericytomas. Moreover, SRF-RELA fusions have been described in a subset of cellular variants of myofibroma and myopericytoma showing myogenic differentiation. Triggered by an index case of an unclassified cellular myoid tumor showing a novel SRF-ICA1L fusion we have investigated our files for cases showing similar histology and screened them using a combined approach of targeted RNA sequencing and fluorescence in situ hybridization. A fusion between SRF exon 4 and ICA1L exon 10 or 11 was identified in a total of 4 spindle cell tumors with similar clinicopathologic features. Clinically, the tumors were deep-seated and originated in the trunk or proximal lower extremity of adult patients (age range: 23 to 55 y). Histologically, the tumors were composed of cellular fascicles of monomorphic eosinophilic spindle cells showing increased mitotic activity, harboring densely hyalinized stroma, often with focal areas of necrosis. All 4 tumors had similar immunoprofiles with positivity for smooth muscle actin, calponin, and smooth muscle myosin heavy chain. Tumors were negative for desmin and caldesmon, markers often seen in SRF-RELA-positive tumors with similar morphology. Follow-up information was available in 3 patients. Two patients had no evidence of disease, 2 and 5 years after surgical resection. One patient, a 35-year-old male patient with a 19 cm deep-seated tumor with brisk mitotic activity (>20 mitoses in 10 HPF), developed lung metastases 7 years after initial diagnosis. In summary, we report a series of 4 cellular myoid tumors with novel SRF-ICA1L gene fusions, characterized by bland spindle cell fascicular growth, expression of specific smooth muscle markers, elevated mitotic activity, marked stromal hyalinization, focal coagulative necrosis, and potential for malignant behavior. Given the morphologic overlap with related cellular myopericytic tumors with SRF-RELA fusions, it is likely that SRF-ICA1L fusions define a similar subset of neoplasms composed of immature smooth muscle cells.

Novel SS18-NEDD4 gene fusion in a primary renal synovial sarcoma.

We report a primary renal synovial sarcoma with a novel gene fusion and unusual morphology. The patient was a 35-year-old female who was found to have a 5 cm hypocellular, myxoid spindle cell renal neoplasm that subtly permeated amongst native renal tubules. The tumor cells showed elongated hyperchromatic nuclei with ill-defined pale cytoplasm, lacking significant mitotic activity or necrosis. Based on its deceptively bland morphology, the differential diagnosis included mainly benign entities, such as metanephric stromal tumor, mixed epithelial stromal tumor (MEST), and myxoid peripheral nerve sheath tumors. A definitive diagnosis of synovial sarcoma was made only subsequently to RNA-sequencing, which revealed a novel SS18-NEDD4 gene fusion. These results were further confirmed by fluorescence in situ hybridization using custom design break-apart probes for both genes. This case illustrates the utility of targeted RNA-sequencing in the classification of challenging tumors with deceptive morphology and identification of novel gene fusion variants. Apart from the canonical SS18-SSX fusion, this is only the second alternative gene fusion variant described in synovial sarcoma to date, in addition to two cases harboring the SS18L1-SSX1 fusion.

Recurrent YAP1 and KMT2A Gene Rearrangements in a Subset of MUC4-negative Sclerosing Epithelioid Fibrosarcoma.

Sclerosing epithelioid fibrosarcoma (SEF) is an aggressive soft tissue sarcoma, characterized by a distinctive epithelioid phenotype in a densely sclerotic collagenous stroma, that shows frequent MUC4 immunoreactivity and recurrent gene fusions, often involving EWSR1 gene. A pathogenetic link with low-grade fibromyxoid sarcoma (LGFMS) has been suggested, due to cases with hybrid morphology as well as overlapping genetic signature. However, a small subset of SEF is negative for MUC4 and lacks the canonical EWSR1/FUS gene rearrangements. Triggered by the identification of recurrent YAP1-KMT2A gene fusions by RNA sequencing in 3 index cases of MUC4-negative, EWSR1/FUS fusion-negative SEF, we further investigated a cohort of 14 similar SEF cases (MUC4-negative, EWSR1/FUS fusion-negative) by fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction, and/or DNA-based massively parallel sequencing (MSK-IMPACT) for abnormalities in these genes. Three additional SEFs with KMT2A gene rearrangements and one additional case with YAP1 gene rearrangements were identified by FISH. In addition, one case with YAP1-KMT2A and one with KMT2A-YAP1 fusion were detected by reverse transcription-polymerase chain reaction and MSK-IMPACT, respectively. As a control group, 24 fibromyxoid spindle cell tumors, diagnosed or suspected as fusion-negative LGFMS, were also tested for YAP1 and KMT2A abnormalities by FISH, but none were positive. The YAP1/KMT2A-rearranged SEF group affected patients ranging from 10 to 86 years old (average and median: 45) of both sexes (4 females, 5 males). The tumors involved somatic soft tissues with a wide distribution, including extremities, trunk, neck, and dura. Histologically, the tumors showed variable cellularity, with monotonous ovoid to epithelioid tumor cells and hyalinized collagenous background typical of SEF. More than half of the cases showed infiltrative borders, within fat or skeletal muscle. No LGFMS component was identified. All tumors were negative for MUC4 and had an otherwise nonspecific immunophenotype. Of the 6 cases with available follow-up information, 2 had local recurrences, and 2 developed soft tissue and/or bone metastases, including 1 of them died of the disease.