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SMARCB1biallelic inactivation resulting in SMARCB1/INI1 deficiency drives a wide range of malignancies, including many mesenchymal tumors. However, the specific types of SMARCB1alterations and spectrum of cooperating mutations among various types of sarcomas has not been well investigated. We profiled SMARCB1genetic alterations by targeted DNA sequencing and fluorescence in situ hybridization (FISH) in a large cohort of 118 soft tissue and bone tumors, including SMARCB1-deficient sarcomas (78, 66%): epithelioid sarcomas, epithelioid peripheral nerve sheath tumors, poorly differentiated chordomas, malignant rhabdoid tumors, and soft tissue myoepithelial tumors, as well as non-SMARCB1-deficient sarcomas (40, 34%) with various SMARCB1genetic alterations (mutations, copy number alterations). SMARCB1 loss by immunohistochemistry was present in 94% SMARCB1pathogenic cases. By combined sequencing and FISH assays, 80% of SMARCB1-deficient tumors harbored homozygous (biallelic) SMARCB1loss, while 14% demonstrated heterozygous SMARCB1loss-of-function (LOF) alterations, and 6% showed no demonstrable SMARCB1alterations. FISH and sequencing were concordant in the ability to detect SMARCB1loss in 48% of cases. Epithelioid sarcomas most commonly (75%) harbored homozygous deletions, while a subset showed focal intragenic deletions or LOF mutations (nonsense, frameshift). In contrast, most soft tissue myoepithelial tumors (83%) harbored SMARCB1nonsense point mutations without copy number losses. Additionally, clinically significant, recurrent co-occurring genetic events were rare regardless of histotype. By sequencing, extended 22q copy number loss in genes flanking the SMARCB1locus (22q11.23) occurred in one-third of epithelioid sarcomas and the majority of poorly differentiated chordomas. Poorly differentiated chordomas and soft tissue myoepithelial tumors showed significantly worse overall and disease-free survival compared to epithelioid sarcomas. Overall, SMARCB1LOF alterations predominate and account for SMARCB1 protein loss in most cases: majority being biallelic but a subset were heterozygous. In contrast, SMARCB1alterations of uncertain significance can be seen in diverse sarcomas types and does not indicate a SMARCB1-deficient entity.

To elucidate the mechanisms underlying the divergent clinicopathologic spectrum of EWSR1/FUS::CREBtranslocation-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/FUSfusion 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, TERTpromoter and CDKN2Amutations were identified only in clear cell sarcoma (CCS) and associated with worse overall survival. CDKN2A/Bhomozygous deletions were recurrent in angiomatoid fibrous histiocytoma (AFH) and restricted to metastatic cases. mRNA upregulation of MITF, CDH19, PARVB, and PFKPwas found in CCS, compared to AFH, and correlated with a hypomethylated profile. In contrast, S100A4and XAF1were 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::CREBtranslocation-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.

Appropriate classification of fusion-driven bone and soft tissue neoplasms continues to evolve, often relying on the careful integration of morphologic findings with immunohistochemical, molecular, and clinical data. Herein, we present 3 cases of a morphologically distinct myxoid mesenchymal neoplasm with myogenic differentiation and novel CRTC1::MRTFB(formerly MKL2) gene fusion. Three tumors occurred in 1 male and 2 female patients with a median age of 72 years (range: 28-78). Tumors involved the left iliac bone, the right thigh, and the left perianal region with a median size of 4.0 cm (4.0-7.6 cm). Although 1 tumor presented as an incidental finding, the other 2 tumors were noted, given their persistent growth. At the time of the last follow-up, 1 patient was alive with unresected disease at 6 months, 1 patient was alive without evidence of disease at 12 months after surgery, and 1 patient died of disease 24 months after diagnosis. On histologic sections, the tumors showed multinodular growth and were composed of variably cellular spindle to round-shaped cells with distinct brightly eosinophilic cytoplasm embedded within a myxoid stroma. One tumor showed overt smooth muscle differentiation. Cytologic atypia and mitotic activity ranged from minimal (2 cases) to high (1 case). By immunohistochemistry, the neoplastic cells expressed focal smooth muscle actin, h-caldesmon, and desmin in all tested cases. Skeletal muscle markers were negative. Next-generation sequencing detected nearly identical CRTC1::MRTFBgene fusions in all cases. We suggest that myxoid mesenchymal tumors with myogenic differentiation harboring a CRTC1::MRTFBfusion may represent a previously unrecognized, distinctive entity that involves soft tissue and bone. Continued identification of these novel myxoid neoplasms with myogenic differentiation will be important in determining appropriate classification, understanding biologic potential, and creating treatment paradigms.

Alveolar rhabdomyosarcoma (ARMS) with FOXO1gene rearrangements is an aggressive pediatric rhabdomyosarcoma subtype that is prognostically distinct from embryonal rhabdomyosarcoma and fusion-negative ARMS. Herein, we report two cases of ARMS with PAX3::MAML3fusions. The tumors arose in an infant and an adolescent as stage IV metastatic disease (by Children’s Oncology Group staging system). Histologically, both cases were small round blue cell tumors arranged in vague nests and solid sheets that were diffusely positive for desmin and myogenin. By methylation profiling and unsupervised clustering analysis, the tumors clustered with ARMS with classic FOXO1rearrangements and ARMS with variant PAX3::NCOA1/INO80Dfusions, but not with biphenotypic sinonasal sarcoma (BSNS) with PAX3::MAML3/NCOA2/FOXO1/YAP1fusions, nor with other small round blue cell tumors, including embryonal rhabdomyosarcoma. The differentially methylated genes between ARMS and BSNS were highly enriched in genes involved in myogenesis, and 21% of these genes overlap with target genes of the PAX3::FOXO1 fusion transcription factor. On follow-up after initiation of vincristine/actinomycin/cyclophosphamide chemotherapy, the tumors showed partial and complete clinical response, consistent with typical upfront chemotherapy responsiveness of ARMS with the classic FOXO1rearrangement. We conclude that PAX3::MAML3is a novel variant fusion of ARMS, which displays a methylation signature distinct from BSNS despite sharing similar PAX3fusions. These findings highlight the utility of methylation profiling in classifying ARMS with non-canonical fusions.

RAD51B-rearranged sarcomas are rare neoplasms that exhibit a heterogeneous morphology. To date, 6 cases have been reported all involving the uterus, including 4 perivascular epithelioid cell tumors (PEComas) and 2 leiomyosarcomas (LMS). In this study, we describe the morphologic, immunohistochemical, and molecular features of 8 additional sarcomas with RAD51B-rearrangement, including the first extrauterine example. All patients were females with a median age of 57 at presentation. Seven tumors originated in the uterus and one in the lower extremity soft tissue, with a median tumor size of 12 cm. Histologically, 4 tumors showed predominantly spindle cell morphology with eosinophilic fibrillary cytoplasm, with or without nuclear pleomorphism, while 2 pleomorphic epithelioid cells, featuring clear to eosinophilic, granular cytoplasm. Two neoplasms exhibited undifferentiated cytomorphology, including one with uniform small blue round cells. All tumors showed high grade cytologic atypia, high mitotic activity (median, 30/10 HPFs), while coagulative necrosis was seen in 6 cases and lymphovascular invasion in 2. By immunohistochemistry, 2 showed myoid and melanocytic markers in keeping with PEComa, while 4 cases were only positive for smooth muscle markers consistent with LMS (including 3 myxoid). The remaining 2 cases had a non-specific immunoprofile. Five cases tested by targeted RNA sequencing (Archer FusionPlex, Illumina TruSight) showed different fusion partners (HMGA2, PDDC1, CEP170). RAD51Brearrangements were identified by FISH in the remaining 3 cases. Targeted DNA sequencing in 2 cases were negative for TSCgene alterations. Clinical outcome available in 5 patients (median follow-up 19 months) revealed 3 local recurrences, 2 lung metastases, and 4 died of disease. Our results expand the spectrum of sarcomas with RAD51B-fusions, demonstrating variable clinical presentations, morphologic spectrum, and fusion partners. These tumors have a predilection for a uterine location, with either LMS, PEComa or undifferentiated phenotypes, and are associated with an aggressive clinical course.