Schoettler, P.J.

ABSTRACT Purpose We studied histologic Ewing sarcoma (ES) tumor response at local control surgery to explore associations between histologic response and chemotherapy practice changes over time and confirm its prognostic significance in the modern era. Methods We reviewed clinical data for 126 patients with ES treated at Mayo Clinic from 1977 to 2022, analyzing the percentage necrosis as a prognostic factor across chemotherapy regimens (including modern interval‐compressed VDC‐IE). Results In the historical cohort, 5‐year event‐free survival (EFS) and overall survival (OS) for patients with ≤5% versus greater than 5% viable tumor were 76% versus 59% ( p = 0.01) and 83% versus 62% ( p = 0.01). Review of 104 specimens by two pathologists identified the percentage remaining viable tumor as more reproducible than the percentage necrosis and/or fibrosis. On multivariable analysis of patients with localized disease ( n = 87), pelvic tumors and greater than 5% viable tumor remained independent risk factors for poor survival (EFS: hazard ratio [HR] 3.1, p < 0.01; OS: HR 2.1, p = 0.048). The modern cohort consisted of 22 patients. No patients with tumor size less than 8 cm at diagnosis and 100% necrosis after surgery had events; four of seven with tumors greater than 8 cm had events despite 100% necrosis. Conclusions Our results validate the historical threshold of greater than 5% viable tumor with older chemotherapy regimens but not with modern interval‐compressed VDC‐IE. In addition, inter‐rater reliability for the percentage viable is more reproducible than the percentage necrosis. Although numbers are small, tumors less than 8 cm with no viable tumor appear to be a favorable subgroup and suggest risk stratification using a combination of tumor size and any viable tumor should be evaluated in ES.

Abstract INTRODUCTION: Pleuropulmonary blastoma (PPB) and Sertoli-Leydig cell tumor (SLCT) are DICER1-related tumors for which novel therapies are urgently needed. A subset of Wilms tumors are also associated with germline or somatic variants in DICER1. Current therapies include cytotoxic chemotherapy, radiation, and surgery, but there are limited options for patients with recurrent disease. Antibody-Drug Conjugates (ADC) have emerged as promising therapies for several solid tumors. Aberrantly expressed cell surface proteins, including Preferentially Expressed Antigen in Melanoma (PRAME), represent potential targets for immunotherapeutic treatments like CAR-T cell and antibody-based therapies in malignant cancers, including DICER1-associated tumors. METHODS: Individuals diagnosed with PPB, SLCT, and Wilms tumor who enrolled in the International PPB/DICER1 Registry, International OTST Registry, and/or UT Southwestern Pediatric Hematology-Oncology Biorepository were selected for analysis. We analyzed ten PPB samples (four PDX, six primary tumor), four SLCT samples (all primary tumor), and 54 Wilms tumor samples (4 PDX, 50 primary tumor). DICER1 hotspot variants were verified in all 14 PPB and SLCT samples, while none of the Wilms tumor samples we analyzed exhibited DICER1 hotspot variants. Mapped and normalized whole-transcriptome sequencing reads were used to examine expression of 73 targets of antibody- or cell-based therapies that are clinically approved or under clinical investigation on clinicaltrials.gov. Genes with a median expression exceeding 2.5 transcripts per million (TPM) were considered expressed in a given tumor type; this threshold was selected to be permissive in identifying potential targets for further evaluation. RESULTS: Of the 73 genes of interest, 21 targetable cell-surface genes met a median expression threshold of 2.5 TPM in at least one tumor type: 11 genes in PPB, 16 in SLCT, and 14 in Wilms. Seven genes (FN1, PRAME, MCAM, CD248, IL11RA, CD276 (B7-H3), and MRC2) were expressed at this level in all three tumor types. Additional genes were expressed above this level in only two of the tumor types; PDGFRA in PPB and SLCT; GPC2 and TYRO3 in SLCT and Wilms tumor; and CSPG4, ERBB2, FZD10 in PPB and Wilms tumor. Six genes were exclusively expressed in SLCT (AXL, GPNMB, PROM1, KDR, ITGA6, FGFR4), and two were unique to Wilms tumor (GPC1, ULBP3). Of these, PRAME was previously reported as frequently expressed in DICER1-associated tumors; little has been reported about the remaining potential targets. CONCLUSION: PPB, SLCT, and Wilms tumor express several potential immunotherapy targets by RNA-seq. Expression of these potential targets should be investigated by immunostaining in a wider panel of tumors. Further study on the potential for ADCs or CAR-T cell therapies to treat DICER1-related conditions is needed. Citation Format: Brenya McNally, BS, Paige H.R. Mallinger, MS, David Huntsman, MD, FRCPC, FCCMG, Yemin Wang, PhD, Joyce Zhang, PhD, Claudette R. Fraire, PhD, Peter J. Schoettler, MD, Kris Ann P. Schultz, MD, Kenneth S. Chen, MD. Identifying potential cell-surface targets for therapeutic treatment of DICER1-related cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr A018.

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 &lt;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 &lt; 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&lt;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&lt;0.001), with AUC 0.988 for distinguishing between ES and HD ENPP1 (p&lt;0.0001). The highest ENPP1 EV values were in patients with large primary tumors (&gt;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.