Bailey, Kelly M.

Pediatric sarcomas are a heterogeneous group of rare mesodermal malignancies. These cancers, which affect children from infancy through adolescence and young adulthood, are in general challenging to treat with currently available therapies. Biologically, many are characterized by quiet genomes, fusion oncoproteins, immune “cold” microenvironments, and vast epigenetic deregulation that contributes to diverse and complex mechanistic drivers. Multifaceted advancements in research strategies, including high-throughput screening, new model systems, surfaceome profiling, and study of oncogenic fusion condensates have led to new opportunities for understanding the biology of pediatric sarcomas. To continue to make progress for these difficult to treat cancers, it will be critical to continue to improve access to bioinformatic data, approach patient care using innovative clinical trial frameworks, and foster interdisciplinary partnerships among medicinal chemists, scientists, clinicians, advocates, and industry partners.

Abstract Background: Pediatric and AYA sarcomas are rare, biologically heterogeneous tumors with limited noninvasive biomarkers to guide diagnosis, personalized treatment, and disease monitoring. Standard-of-care evaluation relies on invasive biopsies that carry procedural risks and imaging modalities with limited sensitivity for detecting minimal residual disease and distinguishing tumor types. Cell-free DNA (cfDNA) methylation profiling offers a minimally invasive alternative capable of capturing tumor-derived epigenomic alterations and has consistently shown strong clinical utility in adult malignancies. However, genome-wide cfDNA methylation patterns across diverse pediatric and AYA sarcoma subtypes remain poorly defined. Methods: Plasma was collected from 93 pediatric and AYA patients (pediatric <18 years; AYA 15-39 years) representing 27 Ewing sarcoma, 17 rhabdomyosarcoma, 11 liposarcoma, 10 chordoma, 8 synovial sarcoma, 7 chondrosarcoma, 6 osteosarcoma, and 6 fibrosarcoma cases, plus 19 matched healthy donors. cfDNA was isolated from 1 mL plasma, quantified by Qubit, and assessed by Bioanalyzer. Libraries were prepared using the NEBNext EM-seq v2 workflow with PCR cycles scaled to cfDNA input amount. Genome-wide sequencing (15× target depth) was followed by alignment, deduplication, and methylation calling. MultiQC was used to assess depth, duplication rate, conversion efficiency, and CpG coverage. Global methylation summaries were computed, and methylKit was used to generate per-CpG and 1-kb tiling matrices and initiate differential methylation analyses. Results: Ewing sarcoma patients had significantly higher plasma cfDNA concentrations than age- and sex-matched healthy donors (24.4 vs 4.6 ng/mL, p < 0.0001), whereas cfDNA levels for other sarcoma subtypes overlapped with the healthy cohort. Low or undetectable cfDNA concentrations by Qubit were far more common among non-Ewing sarcoma samples (23 of 66) than Ewing sarcoma (1 of 27). All samples achieved sufficient CpG coverage for genome-wide methylation modeling. Global methylation summaries revealed no significant differences in mean β-values across sarcoma subtypes or versus healthy donors. An independent analysis of the Ewing subset identified 299 significant differentially methylated regions (DMRs), supporting the detectability of disease-specific methylation changes. Conclusions: These results demonstrate the feasibility of high-quality cfDNA methylome profiling across a large and diverse pediatric and AYA sarcoma cohort, including in plasma samples with cfDNA concentrations below the limit of detection. While global methylation levels appear broadly similar across subtypes, proof-of-concept DMR analysis in Ewing sarcoma suggests that subtype-specific epigenomic signatures could enable future noninvasive diagnostic tools for pediatric and AYA sarcomas. Citation Format: Lilli J. Greiner, Roman O. Kowalchuk, Jeff Szymanski, Pradeep S. Chauhan, Adam Amundson, Peter J. Schoettler, Wendy Allen-Rhoades, Stephanie F. Polites, Peter S. Rose, Kelly Bailey, Linda McAllister-Lucas, Anita Mahajan, Nadia Laack, Aadel Chaudhuri. Defining genome-wide cfDNA methylation landscapes in pediatric and adolescent and young adult (AYA) bone and soft-tissue sarcomas [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 7885.

Abstract Introduction: Detectable circulating tumor DNA (ctDNA) is associated with inferior outcomes in Ewing sarcoma (ES); however, ctDNA clears in nearly all patients within 2 of the 6 chemotherapy cycles before radiotherapy (RT) or surgery. Given a 3-year EFS of 37% on AEWS 1221 (metastatic) and 5-year EFS of 78% on AEWS 1031 (localized), current ctDNA testing fails to predict most relapses. Extracellular vesicles (EVs) are microscopic particles studied as a biomarker in multiple cancer types. We hope to improve the sensitivity of ctDNA in ES and provide the first application of EVs in ES. Methods: cfDNA was isolated from 1mL plasma, quantified by Qubit, and sequenced as NEBNext Enzymatic Methyl-seq v2 libraries. We performed a systematic unsupervised clustering analysis of 27 ES plasma cfDNA profiles using 4 differentially methylated regions (DMR) subsets (63 strong DMRs, top 30, top 15, and 10 promoter-proximal loci), 3 clustering algorithms (k-means, Ward hierarchical, PAM), and k=2-5 clusters, testing associations with tumor burden and progression. EV analysis involved antibody detection against ES surface markers, including LINGO1, ENPP1, CDH11, and CD99. Concentrations of EVs were determined by standardized and calibrated high-resolution flow cytometry then analyzed with custom-built software to automatically determine and apply gates. Samples were de-identified, randomly coded, and run in triplicates. The senior research technologist responsible for EV analysis was blinded to patient information and clinical data. Results: EV analysis included 36 pre-RT samples from 16 metastatic and 20 localized ES patients from a prospectively maintained biobank, with 27 pre-RT samples in the cfDNA analysis and 19 healthy donor (HD) samples. 35 of 36 patients had chemotherapy before sample collection. cfDNA concentrations were markedly elevated in ES patients compared with HD (24.4 vs 4.6 ng/mL, p<0.0001), and 74% of ES samples exceeded the HD 95th-percentile detectability threshold (7.0 ng/mL). Across clustering configurations, ES cfDNA profiles consistently exhibited reproducible methylation substructure. One configuration (PAM clustering, k = 5, 63 strong DMRs) showed a modest but permutation-supported association with primary tumor size (nominal p ≈ 0.04; empirical p ≈ 0.03-0.04). ES samples had increased EV concentration compared to HD (p<0.001), with AUC 0.988 for distinguishing between ES and HD ENPP1 (p<0.0001). The highest ENPP1 EV values were in patients with large primary tumors (>8 cm vs. ≤ 8cm) and metastatic disease (p=0.066). Conclusions: cfDNA- and EV-based liquid biopsy provide encouraging signals for high ES detectability pre-RT. We also show the first study of EVs as a novel biomarker in ES, which may provide complementary information to ctDNA. Multimodal liquid biopsy in ES may identify candidate patients for systemic or local therapy escalation and facilitate personalized care. Citation Format: Roman O. Kowalchuk, Lilli J. Greiner, Yohan Kim, Rubia Noori, Jeffrey Szymanski, William S. Harmsen, Pradeep S. Chauhan, Adam C. Amundson, Andrea M. McMahon, Peter J. Schoettler, Wendy A. Allen-Rhoades, Stephanie F. Polites, Peter S. Rose, Kelly M. Bailey, Linda M. McAllister-Lucas, Anita Mahajan, Fabrice Lucien-Matteoni, Nadia N. Laack, Aadel A. Chaudhuri. Multimodal liquid biopsy of Ewing sarcoma reveals high sensitivity prior to radiotherapy [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 1149.

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.

ABSTRACT Approximately 10% of patients with Ewing sarcoma (EwS) have pathogenic germline variants. Here, we report two cases: first, a novel germline pathogenic variant in partner and localizer of BRCA2 ( PALB2) in a patient with a late EwS relapse. Its impact on homologous recombination is demonstrated, and breast cancer risk is discussed. Second, we report a pathogenic germline variant in RAD50 in a patient with a rare prostate primary EwS and later concurrent relapsed EwS and therapy‐related acute myeloid leukemia (tAML) with two rare KMT2A translocations. Understanding rare pathogenic germline variants and the impact on cancer behavior, response to treatment and treatment‐associated toxicities is important for patients.

Trends in diagnostic biopsy sample collection approaches for primary bone sarcomas have shifted in the past 2 decades. Although open/incisional biopsies used to be the predominant approach to obtain diagnostic material for Ewing sarcoma and osteosarcoma, image-guided core needle biopsies have increased in frequency and are safe for patients. These procedures are less invasive and reduce recovery times but have potential limitations. The quantity and quality of tissue obtained through these procedures vary between institutions. Acquired viable tissue volumes can be low, limiting the conduct of downstream expanded clinical workup, molecular analyses, and research. Patients with advanced Ewing sarcoma and osteosarcoma continue to have overall poor outcomes despite dose-intensive cytotoxic chemotherapy. The biology of treatment resistance is not currently well understood, partly due to limited availability of relevant tissue to study. There is a need for access to quality tumor specimens for molecular and other analyses to identify high-risk tumor subsets and drive discovery to improve patient outcomes. Given broad variability in bone tumor tissue procurement and processing across member institutions, the Children’s Oncology Group Bone Tumor Committee convened a multidisciplinary group of experts to outline the current and near-future tissue needs for optimal clinical care and access to research platforms. The goal of this working group was to provide high-level guidance on biopsy practices that safely meet these evolving needs. Harmonizing tissue collection practices is paramount to improving the care of children, adolescents, and young adults diagnosed with Ewing sarcoma and osteosarcoma.