About

Theodore W. Laetsch, MD, is an Associate Professor of Pediatrics (Oncology) at the Children's Hospital of Philadelphia. He serves as an Attending Physician in the Division of Oncology at the same institution. Dr. Laetsch is the Inaugural Director of the Very Rare Malignant Tumors Program and the Director of the Developmental Therapeutics Program at the Children's Hospital of Philadelphia. He is also a Co-Leader of the Pediatric Oncology Program at the Abramson Cancer Center, University of Pennsylvania. His professional focus includes pediatric oncology, with particular expertise in rare malignant tumors and developmental therapeutics, contributing to clinical care and research in these areas.

Research topics

• Internal medicine
• Oncology
• Medicine
• Immunology
• Biology
• Pediatrics
• Dermatology
• Pathology

Selected publications

• Abstract B008: Personalized antisense oligonucleotide treatment in a patient with relapsed <i>NFIA</i> :: <i>CBFA2T3</i> acute myelogenous leukemia

  Cancer Research · 2026-01-13

  article

  Abstract Introduction Although driver oncogenic fusions offer an appealing therapeutic target, there is a critical shortage of targeted therapies against many fusions. Customizable direct target inhibition would address this gap. We developed an individualized antisense oligonucleotide (ASO) targeting the NFIA::CBFA2T3 fusion for a patient with relapsed acute myelogenous leukemia (AML). Diagnosed at 5 months of age with isolated central nervous system (CNS) AML, the patient underwent surgical resection, radiation therapy, and multiple rounds of systemic and intrathecal (IT)/intraventricular chemotherapy over 18 months. Ultimately, widespread leptomeningeal and extra-CNS AML progression occurred, requiring extraventricular drain (EVD) placement prior to ASO administration. We hypothesized the ASO would eliminate NFIA::CBFA2T3 fusion expression and reduce viability of fusion-bearing cells. Methods CLIA-certified whole transcriptome RNA sequencing was performed at relapse. A panel of five unique ASO constructs were designed against the NFIA::CBFA2T3 fusion breakpoint. Constructs were tested in 3 model cell lines and 1 patient-derived cell line. Neurotoxicity of the lead ASO was evaluated in NOD scid gamma (NSG) mice. A single patient IND application with a dose escalation schema received IRB and FDA approval. GMP-grade ASO (2 mg) was administered via IT injection at disease progression after informed consent was obtained. Adverse events were reported per CTCAE v5.0. Pharmacokinetic analysis on cerebrospinal fluid (CSF) and peripheral blood was performed using liquid chromatography/mass spectrometry. Results The lead ASO diminished NFIA::CBFA2T3 transcript expression by 83% in HEK293T cells expressing NFIA::CBFA2T3 and reduced cell number by 57% (p&amp;lt;0.05) in the patient-derived cell line. There was no decrease of endogenous NFIA mRNA or growth-inhibitory effect to non-fusion-bearing cells. Animal models showed no signs of toxicity. Within 4 months of pre-clinical testing initiation, ASO was administered during a period of rapid disease progression. Surrounding administration, the patient experienced elevated CSF output (maximum 386 mL/day), cerebral edema, and elevated CSF cytokines IL-6 (18-905 pg/mL) and IL-8 (199-4039 pg/mL). Adverse events grade 3 or above with possible or probable attribution to ASO included depressed level of consciousness, cerebral edema, hydrocephalus, and seizure. Maximal ASO CSF concentration was 648 ng/mL at 48 hours and was undetectable by day 5 post ASO. The ASO was not detected in peripheral blood. The patient experienced further AML progression and died 21 days post ASO. Conclusion An NFIA::CBFA2T3 ASO was engineered and demonstrated decreased transcript expression preclinically. The successful clinical delivery demonstrates proof-of-principle for personalized ASOs in pediatric oncologic care. Toxicity attribution is complicated by rapid disease progression. Ongoing work will more deeply phenotype this patient’s clinical course and develop ASO platform trials. Citation Format: Monica Pomaville, Hyojeong Hwang, Alexis Boulter, Tina Glisovic-Aplenc, Praneeth Bommisetti, Katelyn Oranges, Brandi Nelson, Jeffrey Schubert, Feng Xu, Jinhua Wu, Gregory M. Podsakoff, Margaret Tartaglione, Olivia Caradonio, Ellen Maple, Johannes Van Der Loo, Aashim Bhatia, Michael LaRiviere, Madison Hollawell, Mateusz Koptyra, Marilyn Li, Theodore W. Laetsch, Peter Madsen, Jessica B. Foster, Richard Aplenc, Fange Liu. Personalized antisense oligonucleotide treatment in a patient with relapsed NFIA::CBFA2T3 acute myelogenous leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B008.

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• Abstract 1151: Targeting high risk osteosarcoma: MYC modulation alters metastasis

  Cancer Research · 2026-04-03

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  Abstract Osteosarcoma (OS) is a bone tumor that affects human and canine patients. Standard of care is neoadjuvant chemotherapy and surgery resulting in a 5 year survival rate for patients with localized disease of ∼70%. However, patients with metastatic disease and relapsed disease have a 5 year overall survival of less than 30%. Therefore, there is a critical need for improved therapies and a better understanding of the biological underpinnings of high risk disease. A subset of patients with particularly poor outcomes are known to have copy number amplification of MYC. However, it is not known if MYC contributes to the high risk phenotype by driving metastatic progression or drug resistance. Importantly, 20 compounds have been described as MYC inhibitors and perturb different steps of MYC driven transcription. In this report, we found that MYC drives cell migration and outgrowth but does not appear to contribute to drug resistance in OS cells. More precisely, MYC silencing reversed the metastatic phenotypes of migration and outgrowth of OS cells. Further, MYC downstream targets play an important role in metastatic progression. Silencing of MYC in 5 different cell lines revealed 45 common induced targets, many of which are known to modulate different steps in the metastatic cascade. We screened all 20 compounds previously shown to interfere with MYC transcription using an approach designed to capture the compound that modulates both MYC activity and the metastatic phenotype. Fourteen compounds modulated expression of MYC and/or downstream targets in 4 different OS models. Of those,10 compounds had a profound impact in cell viability in both 2D and 3D assays. Five of these showed selective toxicity in 3D relative to 2D; a phenotype linked to metastatic progression. Importantly, not all compounds that modulated MYC showed therapeutically favorable effects on migration or metastatic organization and outgrowth with at least 2 compounds driving a dramatic increase in migration despite suppressing expression of MYC. Nevertheless, 2 compounds, samuraciclib and THZ531, blocked MYC expression, downstream target expression, cell migration, metastatic organization and outgrowth. We confirmed these results and showed reversal of metastatic competence and complete reversal of metastatic outgrowth using the in vivo/ex vivo pulmonary metastasis assay (PuMA). We are now working to integrate CUT&amp;Tag with BRUseq, an assay of nascent transcription, to determine if modulation of different steps in MYC transcription drives diverse cellular phenotypes as we hypothesized. Nevertheless, the top hit of the screen, samuraciclib, convincingly reverses MYC activity and the associated metastatic phenotype and is undergoing additional testing in metastatic OS mouse models and a canine clinical trial is under development. Correlative biology such as spatial transcriptomics will be used to guide the translation of samuraciclib to patients with high risk osteosarcoma. Citation Format: Emily Seiden, Scott Sauer, Emma Hiscock, Nha Nhu Le, Ainsley Hellens, Monica Inda, Andrew Fuller, Sridhar M. Veluvolu, Rachael Hinshaw, Zachary P. Tolstyka, Elissa Levine, Rashmi Chugh, Theodore W. Laetsch, Nouri Neamati, Heather Wilson-Robles, Chand Khanna, David Warshawsky, Patrick J. Grohar. Targeting high risk osteosarcoma: MYC modulation alters metastasis [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 1151.

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• Validation of a Pre-Infusion Prediction Model for IEC-HS in Children and Young Adults with B-Acute Lymphoblastic Leukemia

  Transplantation and Cellular Therapy · 2026-02-01

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  PublisherDOI

• A High‐Sensitivity Circulating Nucleic Acid Sequencing Assay for Assessing Treatment Response to Alectinib in a Pediatric Patient With <i>ALK</i> ‐Rearranged Non–Small Cell Lung Cancer

  Pediatric Blood & Cancer · 2026-04-11

  articleOpen accessSenior author

  To the Editor: Pediatric lung adenocarcinoma is extremely rare, often presents with metastatic disease, and portends a poor prognosis with a median survival of 14 months and a 5-year survival rate of 26% [1-4]. Molecular diagnostics have become essential in pediatric non–small cell lung cancer (NSCLC) management [2, 5-7], including the detection of EGFR, BRAF, and MET mutations and ALK, ROS1, RET, and NTRK translocations [5, 8], enabling the implementation of kinase inhibitors into front-line treatment leading to improved outcomes [5, 8]. Recently, noninvasive “liquid biopsies” for plasma circulating tumor DNA (ctDNA) have been studied as an emerging biomarker in NSCLC that correlates with tumor volume and risk subtype at diagnosis [9]. Although recent studies suggest the potential diagnostic and prognostic value of plasma ctDNA in NSCLC, it has not been incorporated into standardized NSCLC treatment response and surveillance guidelines to inform management decisions due to the heterogeneity of assays used, lack of standardized follow-up timepoints, and suboptimal sensitivity of current assays [10]. A 16-year-old never-smoker girl with no significant past medical history presented to the emergency department with two months of progressive shortness of breath, decreased exercise tolerance, progressively worsening productive cough, intermittent fevers, fatigue, unintentional weight loss, and pleuritic chest pain. Positron emission tomography (PET) demonstrated a hypermetabolic mediastinal mass with contiguous right upper lobe pulmonary involvement encasing the superior vena cava (SVC), with flattening of the distal trachea and narrowing of the right mainstem bronchus, and multiple hypermetabolic right upper lobe pulmonary lesions in addition to hypermetabolic osseous, hepatic, and local and distant lymph node metastases (Figure 1A–C). Chest computed tomography (CT) demonstrated a mediastinal mass with mass effect on the trachea, SVC, and right mainstem bronchus with diffuse lymphadenopathy, in addition to hepatic hypodensities and heterogeneous appearance of the bone marrow at the vertebral body of T7. There was no evidence of metastatic brain lesions on the brain MRI. A left supraclavicular lymph node was biopsied and demonstrated portions of partially encapsulated fibrous tissue nearly entirely involved by a proliferation of medium to large malignant-appearing cells. Lesional cells had abundant pale mucinous to eosinophilic cytoplasm with large nuclei and prominent nucleoli growing in sheets and nests, and in many areas, also showed micropapillary formation (Figure 2A). Immunostaining performed across multiple institutions showed tumor cells in the lymph node to be positive for pancytokeratin, CK7, TTF-1, and Napsin A, with diffuse weak staining for ALK (D5F3 clone), and the presence of intracytoplasmic mucin was confirmed by mucicarmine cytochemical staining (Figure 2B–E). Overall, the morphology and immunoprofile with radiologic imaging data were consistent with a diagnosis of stage IV moderately differentiated lung adenocarcinoma. The RNA fusion panel of the metastatic supraclavicular lymph node lesion demonstrated an EML4::ALK fusion. Given the detectable EML4::ALK fusion, the patient was started on alectinib 600 mg BID, as alectinib has demonstrated superior CNS disease-free survival compared to platinum-based chemotherapy [11] and superior progression-free survival, lower rates of CNS progression, and reduced toxicity compared to crizotinib in previously untreated adults with advanced stage ALK-rearranged NSCLC, including patients diagnosed with stage IV disease [12]. Two weeks after initiation of alectinib therapy, the EML4::ALK fusion was detected by Caris Assure circulating nucleic acid sequencing (cNAS; Supplementary Table S1). The patient remained adherent to alectinib and interval chest computed tomography (CT) scans and whole-body PET scans obtained at 2 and 4 months after starting alectinib therapy demonstrated a partial response (PR) in pulmonary and metastatic disease as per RECIST criteria (at least 30% decrease in sum of diameters of target lesions with no new target lesions) and brain MR continued to be negative for metastatic brain lesions. After 8.5 months of alectinib therapy, chest CT and whole-body PET continued to show an improved PR as per RECIST criteria with no evidence of pulmonary disease and with a residual hypermetabolic hepatic focus (Figure 1D–F). At the same time point, Caris Assure cNAS testing showed loss of EML4::ALK fusion detection (Supplementary Table S1). After 14 months of alectinib therapy, chest CT and whole-body PET demonstrated a complete response (CR) as per RECIST criteria (disappearance of all target lesions and pathological lymph nodes <10 mm in short axis; Figure 1G–I) and Caris Assure cNAS continued to demonstrate loss of EML4::ALK fusion detection (Supplementary Table S1). The high-sensitivity plasma cNAS (Caris Assure) assay used in this report addresses key barriers to ctDNA analysis as a standardized approach for targeted therapy selection and response monitoring, including correlation of radiographic response with loss of EML4::ALK fusion detection on cNAS. Analysis of both cell-free DNA and RNA from the plasma enhances the detection of clinically relevant alterations, and incorporation of paired white blood cell (WBC) sequencing enables discrimination of true tumor-derived variants from false positives, such as those arising from clonal hematopoiesis (CH), ultimately improving the reliability and clinical interpretability of results [10, 13]. This cNAS method enables comprehensive whole-exome and transcriptome analysis of tumor-derived alterations, including detection of de novo and compound mutations in oncogenic drivers, kinase fusions, and alterations in off-target pathways [13]. This allows for the identification of resistance mechanisms to specific kinase inhibitors prior to the onset of symptoms or radiographic evidence of progression or relapse [13]. This report supports the need for further studies to evaluate how detection and subsequent loss of oncogenic drivers with a high-sensitivity cNAS assay correlates with radiographic response to targeted therapy at standardized intervals in receptor tyrosine kinase (RTK)-mutated and kinase fusion-driven pediatric malignancies. The authors confirm that there are no conflicts of interest. The authors confirm that written consent for submitting and publishing this case report, including images and associated text, has been obtained from the patient's family. There are no identifiers included in this report. A.D.G. is supported by NIH grant 2T32CA009615, and T.W.L. is supported by the Alex's Lemonade Stand Foundation Center of Excellence and NIH grant 1R50CA305079. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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• Molecular Characterization of a Complex <i>PDGFRB</i> Structural Variation in Infantile Myofibroma With Complete Response to Imatinib

  JCO Precision Oncology · 2026-01-01

  articleOpen access
  PublisherOA PDFDOI

• 209P Efficacy and safety of larotrectinib as first-line treatment for patients with TRK fusion cancer

  ESMO rare cancers. · 2026-03-01

  articleOpen access

  Methods:We first established patient-derived sarcoma models that recapitulate patient tumor biology.We performed whole-genome CRISPR/Cas9 knockout screens in ex vivo myxofibrosarcoma and dedifferentiated chondrosarcoma cell models.In parallel, these models underwent customized drug-library screening using 82 clinically relevant or preclinically evaluated compounds in a sarcosphere format.Targets of interest identified from both screens were validated across 10 ex vivo sarcoma models and the underlying molecular mechanism was investigated.Patient-derived xenograft (PDX) validation is currently underway.Results: By integrating results from genetic and pharmacological screens, we identified the neddylation pathway as a selective dependency in sarcomas with HRD.Inhibition of the NEDD8-activating enzyme (NAE) with pevonedistat triggered the activation of the unfolded protein response (UPR) pathway and ultimately apoptosis specifically in cell models with HRD.This effect was accompanied by induction of CHOP and NOXA and was recapitulated by genetic disruption of the pathway.Combination screens further revealed strong drug synergy between pevonedistat and inhibitors of the DNA damage response. Conclusions:We found that sarcoma cell models with a high level of HRD were selectively sensitive to neddylation inhibition, highlighting a potential therapeutic niche for patient stratification and future clinical applications.

  PublisherOA PDFDOI

• Abstract 643: The Sean Karl Cohort: An international single-cell RNAseq study of paired patient Ewing sarcoma specimens.

  Cancer Research · 2026-04-03

  article

  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.

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• Abstract A029: Larotrectinib long-term efficacy and safety in pediatric patients with TRK fusion non-primary CNS tumors: Analysis update

  Cancer Research · 2026-01-13

  article

  Abstract Background: NTRK gene fusions are oncogenic drivers across various pediatric and adult tumor types. The prevalence of NTRK gene fusions varies widely, from high (up to 90%) in rare tumors such as infantile fibrosarcoma and secretory carcinoma of the breast to low (&amp;lt;0.5%) in common cancers like non-small cell lung and colorectal carcinoma. Larotrectinib is the first-in-class, highly selective, central nervous system (CNS)-active TRK inhibitor approved for tumor-agnostic use in patients with TRK fusion cancer based on a robust and durable objective response rate in both adult and pediatric patients with various tumor types. Here, we report updated data on larotrectinib-treated pediatric patients with TRK fusion non-primary CNS tumors. Methods: This analysis included patients from 2 clinical trials (NCT02637687 [SCOUT], NCT02576431 [NAVIGATE]). Responses were independent review committee-assessed (Response Evaluation Criteria in Solid Tumors [RECIST] v1.1). In SCOUT, patients could stop larotrectinib in the absence of on-treatment progression (“wait-and-see”). Responses in patients who were re-treated due to progression were assessed by investigators (RECIST v1.1). Results: Ninety-nine patients with non-primary CNS tumors were eligible for analysis as of July 2024, including 49% with infantile fibrosarcoma, 41% with soft tissue sarcoma, and 9% with other solid tumors. Overall response rate was 86% (95% confidence interval [CI] 77–92). In total, 53 patients had complete responses (CR; including 17 pathological CR), 32 had partial responses (PR), 9 had stable disease (SD), and 3 had progressive disease (PD); responses were undefined in 2 patients. Median time to response was 1.8 months (range 0.9–7.3). Median duration of response was 51 months (95% CI 31-not estimable [NE]). Median progression-free survival and overall survival (OS) were 49 months (95% CI 32–NE) and not reached (NR), respectively. The 5-year OS rate was 87% (95% CI 80–95). Median time to investigator-assessed treatment failure (from larotrectinib initiation to earliest documented on-treatment disease progression, start of other anticancer treatment, or death) was NR. Of 54 patients who entered a first “wait-and-see” period (median duration 33 months [range 1–72]), 18 resumed treatment due to PD. Of these, 11 had a response (6 CR and 5 PR [including 2 pending confirmation]), 5 had SD, 1 was not evaluable, and 1 was undefined. Most treatment-related adverse events (TRAEs) were Grade 1/2. Three patients (3%) discontinued due to a TRAE. Conclusions: Larotrectinib demonstrated rapid and durable responses, extended survival, and favorable safety in pediatric patients with TRK fusion cancer. This supports the wider adoption of next-generation sequencing panels that include NTRK gene fusions to identify pediatric patients who may benefit from targeted treatment. Citation Format: Leo Mascarenhas, Theodore W. Laetsch, Birgit Geoerger, Steven G. DuBois, Miranda P. Dierselhuis, Catherine M. Albert, Claudia Blattmann, Helen Toledano, Noah Federman, Ramamoorthy Nagasubramanian, Alberto Pappo, Tanya Watt, Domnita-Ileana Burcoveanu, Esther De La Cuesta, Natascha Neu, Daniel H. Orbach, Yizhuo Zhang. Larotrectinib long-term efficacy and safety in pediatric patients with TRK fusion non-primary CNS tumors: Analysis update [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A029.

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• Trabectedin and low-dose irinotecan to target EWS::FLI1 in Ewing sarcoma: a phase 1/2 trial

  Nature Medicine · 2026-04-16

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  PublisherDOI

• Abstract A016: ews-nf: A custom workflow for tumor cell annotation and analysis of single-cell RNA-sequencing of paired patient Ewing sarcoma specimens from the Sean Karl cohort

  Cancer Research · 2025-09-25

  article

  Abstract Ewing sarcoma (EwS) is a fusion oncoprotein-driven primary bone cancer that demonstrates vast intra- and inter-tumoral heterogeneity. Tumor cell subpopulations, tumor progression, and therapeutic vulnerabilities of cell subpopulations are poorly understood. Paired patient samples (primary and metastatic disease or relapse) are rare at any one institution, and collaborative efforts are needed to address these pressing biologic questions. A national collaborative effort (the Sean Karl cohort) has been established to conduct single-cell RNAseq analyses of retrospective paired tumor samples from patients with EwS and serial samples due to metastasis or relapse using the GEM-X Flex Gene Expression protocol from 10x Genomics. Four analytic teams from multiple institutions will be performing custom downstream analyses to understand the therapeutic vulnerabilities of EwS cell subsets and discern immunobiologic dysfunction. To ensure reproducibility, all data pre-processing and common analyses, such as cell type annotation, are centralized using reproducible workflows developed by the Childhood Cancer Data Lab, a program of Alex’s Lemonade Stand Foundation. Gene expression is quantified using an open-source workflow, scpca-nf. The output from scpca-nf, which includes raw and normalized gene expression, dimensionality reduction, and annotation of non-malignant cells, is used as input to a custom Nextflow workflow, ews-nf, to annotate and analyze tumor cells in Ewing sarcoma samples. Tumor cells are annotated using two complementary methods: AUCell is used to evaluate expression of EwS-specific gene sets, and inferCNV is used to obtain a CNV profile for each cell by comparing potentially malignant cells to definitively non-malignant cells (e.g., immune cell types). Cells with high expression of EwS-specific gene sets and high CNV profiles, relative to immune cell types, are annotated as tumor cells. Tumor cells are further divided into EWS::FLI1 “low” and “high” cells based on expression of custom gene sets. All tumor cells are then analyzed using non-negative matrix factorization to identify and label recurrent gene expression programs found across all samples in the cohort. Processing and sequencing of samples are ongoing at the time of abstract submission. Ultimately, the output from ews-nf will be used to create a harmonized dataset to be shared with all four analytic teams. This harmonized dataset will contain the processed gene expression data, labeling of tumor cells and tumor cell states, and identification of recurrent gene expression programs. This enables all analytical teams to conduct downstream analysis using the same set of tumor cell annotations, making it easy for teams to compare results and draw conclusions. After completion of the study, the ews-nf workflow will be made publicly available to the research community. The processed gene expression data from the Sean Karl cohort, including the tumor cell annotations, will also be made available on the Single-cell Pediatric Cancer Atlas Portal for others to use in their own research. Citation Format: Allegra G Hawkins, Stephanie J Spielman, Joshua A Shapiro, Abbe Pannucci, Elina Mukherjee, Jessica Daley, Shireen Ganapathi, Elissa Boguslawski, Lea F Surrey, Patrick Azar, Filemon Dela Cruz, Jovana Pavisic, Emily Stockfisch, Azfar Neyaz, Ivy John, Jennifer Picarsic, Yutaro Tanaka, Riaz Gillani, Katherine A Janeway, Jaclyn N Taroni, Jessica Davis, Damon Reed, Adam Shlien, Theodore Laetsch, Rajen Mody, Elizabeth R Lawlor, Patrick Grohar, Anthony R Cillo, Kelly M Bailey. ews-nf: A custom workflow for tumor cell annotation and analysis of single-cell RNA-sequencing of paired patient Ewing sarcoma specimens from the Sean Karl cohort [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A016.

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Recent grants

• Image-guided doxorubicin delivery for pediatric sarcomas

  NIH · $2.8M · 2015–2022

Frequent coauthors

• Cornelis M. van Tilburg

  Hopp Children's Cancer Center Heidelberg

  246 shared

• Muna Qayed

  Aflac (United States)

  190 shared

• Michael A. Pulsipher

  159 shared

• Stephan A. Grupp

  Children's Hospital of Philadelphia

  157 shared

• Alexander Drilon

  138 shared

• Michael R. Verneris

  Gates (United States)

  127 shared

• Birgit Geoerger

  Institut Gustave Roussy

  122 shared

• Douglas S. Hawkins

  University of Washington

  113 shared

Labs

• Theodore W. Laetsch LaboratoryPI