John, Ivy

Abstract Introduction: Ewing sarcoma (EwS) is a FET::ETS family member-driven primary bone cancer demonstrating vast heterogeneity. Between a patient’s primary diagnosis and disease progression, tumor cell state adaptation, microenvironment changes, and the landscape of evolving therapeutic vulnerabilities remain poorly understood. To address these gaps, the Sean Karl Cohort was established in 2025 to conduct the largest single-cell transcriptomic analysis of retrospective paired tumor samples from patients with EwS. Here, we present data from the five analytic teams, and through shared SOPs now expand this cohort to Europe. Methods: Common sample processing SOPs were established to isolate cells from FFPE material from paired patient EwS samples. Single-cell (sc) RNAseq was generated using the GEM-X Flex Gene Expression protocol (10x Genomics). Alex’s Lemonade Stand Foundation (ALSF) Data Lab established a common data processing and integration pipeline with an EwS-specific cell annotation workflow to create a harmonized dataset for downstream analyses. Analytic teams were created to define tumor cell subpopulations and their therapeutic vulnerabilities and to characterize the tumor microenvironment. Data: We demonstrate the feasibility of generating high-quality scRNAseq profiles from retrospective FFPE-preserved EwS tumors. To date, 116 samples have yielded ∼800,000 cells. Integrated analysis reveals reproducible EwS tumor cell states shared across patients with regulatory network analyses nominating candidate therapeutic vulnerabilities. The scale of this international cohort enables saturation analysis for rare cell populations. Paired and longitudinal samples further allow correlation of emergent cell states with therapy resistance and metastatic progression. For example, immune-focused analyses show increases in T cell and macrophage populations in post-therapy samples. A novel, standardized EwS-specific cell annotation workflow has been developed to harmonize analyses and findings. These data will be openly shared with the community through the ALSF Single-cell Pediatric Cancer Atlas Portal. Conclusion: Large-scale international collaborative sample sharing, standardized processing pipelines, and harmonized EwS-specific annotations now enable deep single-cell analyses to characterize tumor heterogeneity in this rare pediatric and adolescent cancer. An in vivo expansion of the Sean Karl Cohort is underway to assess conservation of EwS tumor cell states and regulatory vulnerabilities identified in human tumors pre- and post-therapy in preclinical models, further supporting translation of these findings toward therapeutic strategies. Citation Format: Abbe Pannucci, Elina Mukherjee, Jessica D. Daley, Shireen Sita Ganapathi, Elissa Boguslawski, Lea Surrey, Lauren Gutstein, Patrick Azar, Emily Stockfisch, Azfar Neyaz, Ivy John, Jennifer Picarsic, Yutaro Tanaka, Byron Butaney, Riaz Gillani, Brian D. Crompton, Katherine A. Janeway, Jaclyln Taroni, Jessica Davis, Damon Reed, Adam Shlien, Theodore W. Laetsch, Rajen Mody, Clémence Henon, Thomas G. Grünewald, Elizabeth R. Lawlor, Filemon Dela Cruz, Patrick J. Grohar, Jovana Pavisic, Ally Hawkins, Anthony R. Cillo, Kelly M. Bailey. The Sean Karl Cohort: An international single-cell RNAseq study of paired patient Ewing sarcoma specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 643.

Pleomorphic liposarcoma (PLPS) is a highly aggressive sarcoma comprising variable numbers of pleomorphic lipoblasts mixed with undifferentiated pleomorphic sarcoma (UPS)-like areas. Morphologic variants, such as myxofibrosarcoma-like or epithelioid, may cause diagnostic confusion, especially on a core biopsy, but there are few data on the prognostic significance of these features. A total of 120 PLPS biopsies and resection specimens were reviewed and catalogued based on the presence of myxofibrosarcoma-like, UPS-like, and epithelioid foci, in 10% increments. The clinical parameters were collected. Cases occurred in 75 males and 45 females, ranging from 8 to 98 years (median, 62.5 y). Cases arose in the extremities (n=72), trunk (n=32), head/neck (n=10), bone (n=4), mediastinum (n=1), or viscera (colon polyp, n=1). Of those with known depth (n=81), 40 were intramuscular, 34 were subcutaneous, and 7 arose in the dermis. Their sizes ranged from 1 to 24.5 cm (median, 7 cm). Of the patients with ≥1 month of follow-up (n=70), 5 had recurrence and 15 had metastasis. The 5-year overall survival and event-free survival rates were 66.2% and 63.1%, respectively. Tumors ≥5 cm had inferior overall survival compared with tumors <5 cm. The presence of epithelioid areas was also statistically significant in terms of poorer overall survival and event-free survival, while tumors with ≥50% undifferentiated pleomorphic-like areas had better overall survival. There was a trend towards poorer outcomes in tumors with necrosis (≥1%). PLPS is an aggressive adipocytic malignancy that is most commonly present in the extremities of older adults. The morphologic features of these tumors are diverse, and they may be mistaken for UPS or myxofibrosarcoma, carcinoma, and melanomas, particularly on biopsies. Tumor size, necrosis, and epithelioid morphology are associated with adverse prognosis.

AbstractA novel group of S100‐ and CD34‐positive spindle cell tumors with distinctive stromal and perivascular hyalinization harboring recurrent gene fusions involving kinases includingRAF1,BRAF,NTRK1/2/3, andREThave been recently reported. To our knowledge, no such cases harboringALKrearrangements have been identified. We report a previously healthy 41‐year‐old male with a 12‐cm intramuscular shoulder mass. The tumor was composed of bland‐appearing spindled to epithelioid cells, arranged in a patternless pattern in a background of loose myxoid stroma containing striking amianthoid‐like stromal collagen and perivascular rings. In accordance with the previously reported tumors, the tumor cells showed diffuse immunopositivity with S100 and CD34, while lacking SOX10 expression. Targeted RNA‐based next‐generation sequencing identified a novel serine/threonine‐protein phosphatase PP1‐beta‐catalytic subunit (PPP1CB)‐ALKfusion gene. Although ALK break‐apart was not detected by FISH, likely due to a paracentric inversion of chromosome 2, the presence of the fusion was confirmed by Sanger sequencing showing a 10‐bp linker between exon 6 ofPPP1CBand intron 19 ofALKwhile maintaining reading frame. Subsequent ALK‐1 immunostain exhibited diffuse cytoplasmic staining in the tumor cells. Our case expands the molecular genetic spectrum of the distinctive group of spindle cell tumors with CD34/S100+ immunophenotype, supporting the important role of various kinases as drivers of oncogenesis. Awareness of this entity including its unique morphologic and immunophenotypic features as well as its interchangeable kinase gene fusions is crucial for correct classification and potential targeted therapy, particularly in aggressive subsets.

Aims Limited data exist on atypical lipomatous tumour (ALT)/well‐differentiated liposarcoma (WDL) and de‐differentiated liposarcoma (DDLPS) in children and young adults. Methods and results Cases of ALT/WDL/DDLPS arising in patients aged ≤ 40 years were collected from multiple institutional and consultation archives. A total of 116 cases of ALT/WDL (75) and DDLPS (41) were identified, representing fewer than 5% of these tumours seen at our institutions during this time‐period. The patients (59 male/57 female) ranged in age from 8 to 40 years. Sites included deep central (abdomen/retroperitoneum/pelvis/groin) ( n = 60), extremity ( n = 42), trunk ( n = 5), head/neck ( n = 8) and mediastinum ( n = 1). De‐differentiated patterns included: high‐grade pleomorphic sarcoma, myxofibrosarcoma‐like, heterologous rhabdomyoblastic differentiation, low‐grade spindle cell sarcoma and homologous lipoblastic differentiation. Forty‐one patients experienced a local recurrence and 11 patients with DDLPS developed metastasis. ALT arising in the extremities had lower recurrence rates than deep central WDL (5‐year recurrence‐free survival 88.9% versus 59.0%; P = 0.002), while patients with deep central DDLPS experienced significantly more adverse events compared to WDL at this site (5‐year event‐free survival 11.9% versus 59.0%) ( P < 0.0001). Seven (of eight) head/neck tumours had follow‐up available; five recurred, and one patient (DDLPS) with recurrence also experienced a metastasis. The single mediastinal tumour (DDLPS) recurred and metastasised. Conclusion ALT/WDL and DDLPS occurring in patients aged ≤ 40 years is rare, but exhibits similar morphological features to its counterparts in older adults, including potential for heterologous and homologous de‐differentiation in the latter. Although case numbers are limited, tumours arising in the head and neck exhibit high rates of adverse events, suggesting that classification as WDL rather than ALT is more appropriate.

AimsChondroblastomas (CB) are rare bone tumours that typically arise in the epiphysis/apophysis of long bones in skeletally immature patients. We explore the clinicopathological features of CB presenting in adults.Methods and resultsCB in patients ≥20 years of age were retrieved from our institutional archives. Thirty‐nine CB were identified (29 male/10 female; aged 20–54 years). Twenty (51%) cases occurred in long tubular bones, 10 (26%) in small bones of the feet, five (13%) in flat bones and four (10%) in the patella. All cases showed classic cytological features of CB, and chondroid matrix was universally present. Calcification was identified in 10 cases (26%), including various combinations of serpiginous (n = 7), punctate (n = 6), classic chicken‐wire (n = 4) and psammomatous (n = 2) patterns. Haemosiderin (n = 19), woven bone (n = 13), secondary aneurysmal bone cyst formation (n = 8), foamy macrophages (n = 4), hyalinised vascular spaces (n = 2) and cholesterol clefts (n = 2) were noted. Follow‐up information (n = 32, 1–452 months) revealed local recurrence in three patients, all >40 years of age with flat bone origin, one of which developed pulmonary metastases 132 months after initial diagnosis.ConclusionsCB in patients >20 years of age more frequently involves the short bones of the hands/feet and flat bones compared to those arising in their younger counterparts. A subset may harbour extensive serpiginous or psammomatous calcification rather than the classic chicken‐wire pattern. Although the overall local recurrence rate in adulthood is approximately 10%, all three patients with recurrent disease had tumours involving flat bones, suggesting that tumours arising in these sites may behave more aggressively.