Novel PAX3::MAML3 fusion identified in alveolar rhabdomyosarcoma, using DNA methylation profiling to expand the genetic spectrum of "fusion-positive" cases.

Alveolar rhabdomyosarcoma (ARMS) with FOXO1 gene 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::MAML3 fusions. 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 FOXO1 rearrangements and ARMS with variant PAX3::NCOA1/INO80D fusions, but not with biphenotypic sinonasal sarcoma (BSNS) with PAX3::MAML3/NCOA2/FOXO1/YAP1 fusions, 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 FOXO1 rearrangement. We conclude that PAX3::MAML3 is a novel variant fusion of ARMS, which displays a methylation signature distinct from BSNS despite sharing similar PAX3 fusions. These findings highlight the utility of methylation profiling in classifying ARMS with non-canonical fusions.

EWSR1::ATF1 fusions characterize a group of extra-abdominal epithelioid and round cell mesenchymal neoplasms, phenotypically overlapping with sclerosing epithelioid fibrosarcomas, and intra-abdominal FET::CREB fusion neoplasms.

With the increasing use of next generation sequencing in soft tissue pathology, particularly in neoplasms not fitting any World Health Organization (WHO) category, the spectrum of EWSR1 fusion-associated soft tissue neoplasms has been expanding significantly. Although recurrent EWSR1::ATF1 fusions were initially limited to a triad of mesenchymal neoplasms including clear cell sarcoma of soft tissue, angiomatoid fibrous histiocytoma and malignant gastrointestinal neuroectodermal tumor (MGNET), this family has been expanding. We herein describe 4 unclassified extra-abdominal soft tissue (n = 3) and bone (n = 1) neoplasms displaying epithelioid and round cell morphology and carrying an EWSR1::ATF1 fusion. Affected were 3 males and 1 female aged 20-56 years. All primary tumors were extra-abdominal and deep-seated (chest wall, mediastinum, deltoid, and parapharyngeal soft tissue). Their size ranged 4.4-7.5 cm (median, 6.2). One patient presented with constitutional symptoms. Surgery with (2) or without (1) neo/adjuvant therapy was the treatment. At last follow-up (8-21 months), 2 patients developed progressive disease (1 recurrence; 1 distant metastasis). The immunophenotype of these tumors is potentially misleading with variable expression of EMA (2 of 3), pankeratin (2 of 4), synaptophysin (2 of 3), MUC4 (1 of 3), and ALK (1 of 3). All tumors were negative for S100 and SOX10. These observations point to the existence of heretofore under-recognized group of epithelioid and round cell neoplasms of soft tissue and bone, driven by EWSR1::ATF1 fusions, but distinct from established EWSR1::ATF1-associated soft tissue entities. Their overall morphology and immunophenotype recapitulate that of the emerging EWSR1/FUS::CREB fusion associated intra-abdominal epithelioid/round cell neoplasms. Our cases point to a potentially aggressive clinical behavior. Recognizing this tumor type is mandatory to delineate any inherent biological and/or therapeutic distinctness from other, better-known sarcomas in the differential diagnosis including sclerosing epithelioid fibrosarcoma.

ALK-rearranged, CD34-positive spindle cell neoplasms resembling dermatofibrosarcoma protuberans: a study of seven cases.

AIMS: The majority of dermatofibrosarcoma protuberans (DFSP) harbour PDGFB or PDGFD rearrangements. We encountered ALK expression/rearrangement in a PDGFB/D-negative CD34-positive spindle cell neoplasm with features similar to DFSP, prompting evaluation of ALK-rearrangements in DFSP and plaque-like CD34-positive dermal fibroma (P-LDF). METHODS AND RESULTS: We searched the archives of academic institutions for cases previously coded as DFSP and P-LDF. NGS-naïve or PDGFB-negative DFSP were screened for ALK (clone D5F3) expression by immunohistochemistry. NGS or ALK FISH was performed on ALK-positive cases. Methylome profiling studies were performed and compared with conventional DFSP. One case of "DFSP" and two "P-LDF" with ALK expression were identified from the archives, while four cases were detected prospectively. These seven cases (6F:1M; 8 months to 76 years) arose in the dermis of the arm (two), scalp, eyelid, thigh, abdomen, and shoulder and ranged from 0.4 to 4.2 cm. Tumours were composed of spindled cells and displayed a storiform growth pattern. Cytologic atypia was absent, and mitotic figures were scarce (0-2/10 HPFs, high power fields). The lesional cells were diffusely positive for CD34 and ALK and negative for S100 protein. By NGS (n = 5), ALK fusion partners included DCTN1 (2), PLEKHH2, and CLIP2 in DFSP-like cases and FLNA in P-LDF-like lesions. ALK FISH was positive in one (of two) cases previously labelled P-LDF. Methylome profiling of two (of three) ALK-rearranged DFSP-like tumours showed clustering with conventional DFSP in the UMAP dimension reduction plot. To date, no tumour has recurred (n = 2; 26, 27 months). CONCLUSION: We describe a cohort of novel ALK-rearranged tumours with morphologic features similar to DFSP.

Myoepithelial tumors of soft tissue and bone in children and young adults: A clinicopathologic study of 40 cases occurring in patients ≤ 21 Years of age.

Myoepithelial tumors of the soft tissue and bone occurring in patients 21 years of age and younger are rare, and their clinicopathologic features remain incompletely understood. We studied a well-characterized series of 40 such tumors. Cases were retrieved from our archives for the period 2009-2022 and re-reviewed. Available immunohistochemical and molecular genetic data was collected. Clinical information including available follow-up was obtained. The tumors occurred in 18 males and 22 females, ranging from 3 months to 21 years of age (median 11.5 years), and involved a wide variety of soft tissue (n = 36) and bone (n = 4) locations. Histologically benign myoepithelial tumors tended to occur in adolescents (median age 14.5 years; range 5-21 years), whereas myoepithelial carcinomas occurred in younger patients (median age 8.5 years; range 3 months-20 years). Microscopically, the tumors showed a complex admixture of epithelioid, plasmacytoid and spindled cells in a variably hyalinized, myxoid, chondroid or chondromyxoid background. Small subsets of histologically malignant tumors had rhabdoid or "round cell" features. Immunohistochemistry showed 35/40 (88%) cases to be positive with at least one keratin antibody. The 5 keratin-negative tumors were uniformly positive for S100 protein and/or SOX10 and expressed EMA (4 cases) and/or p63 (3 cases). EMA, SMA and GFAP were positive in 21/25 (84%), 13/21 (62%), and 8/21 (38%) tumors, respectively. SMARCB1 and SMARCA4 expression was retained in 29/31 (94%) and 22/22 (100%) of cases, respectively. FISH for EWSR1 gene rearrangement was positive in 6/18 (33%) tested cases. Two EWSR1-negative tumors were also FUS-negative. NGS identified EWSR1::POU5F1, FUS::KLF17, and BRD4::CITED1 gene fusions in 3 tested cases. Clinical follow-up (22 patients; median 23 months; range 1-119 months) showed 3 patients with local recurrences and 5 with distant metastases (lymph nodes, lung, and brain). Three patients died of disease, 3 were alive with recurrent or unresectable disease, and 16 were disease-free. Adverse clinical outcomes were seen only in patients with malignant tumors. We conclude that myoepithelial neoplasms of soft tissue and bone are over-repesented in patients ≤21 years of age, more often histologically malignant, and potentially lethal. Histologic evaluation appears to reliably predict the behavior of these rare tumors.

Novel CRTC1::MRTFB(MKL2) Gene Fusion Detected in Myxoid Mesenchymal Neoplasms With Myogenic Differentiation Involving Bone and Soft Tissues.

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::MRTFB gene fusions in all cases. We suggest that myxoid mesenchymal tumors with myogenic differentiation harboring a CRTC1::MRTFB fusion 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.