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PURPOSE Novel biomarkers are needed to differentiate outcomes in intermediate-risk rhabdomyosarcoma (IR RMS). We sought to evaluate strategies for identifying circulating tumor DNA (ctDNA) in IR RMS and to determine whether ctDNA detection before therapy is associated with outcome. PATIENTS AND METHODS Pretreatment serum and tumor samples were available from 124 patients with newly diagnosed IR RMS from the Children's Oncology Group biorepository, including 75 patients with fusion-negative rhabdomyosarcoma (FN-RMS) and 49 with fusion-positive rhabdomyosarcoma (FP-RMS) disease. We used ultralow passage whole-genome sequencing to detect copy number alterations and a new custom sequencing assay, Rhabdo-Seq, to detect rearrangements and single-nucleotide variants. RESULTS We found that ultralow passage whole-genome sequencing was a method applicable to ctDNA detection in all patients with FN-RMS and that ctDNA was detectable in 13 of 75 serum samples (17%). However, the use of Rhabdo-Seq in FN-RMS samples also identified single-nucleotide variants, such as MYOD1 L122R, previously associated with prognosis. Identification of pathognomonic translocations between PAX3 or PAX7 and FOXO1 by Rhabdo-Seq was the best method for measuring ctDNA in FP-RMS and detected ctDNA in 27 of 49 cases (55%). Patients with FN-RMS with detectable ctDNA at diagnosis had significantly worse outcomes than patients without detectable ctDNA (event-free survival, 33.3% v 68.9%; P = .0028; overall survival, 33.3% v 83.2%; P < .0001) as did patients with FP-RMS (event-free survival, 37% v 70%; P = .045; overall survival, 39.2% v 75%; P = .023). In multivariable analysis, ctDNA was independently associated with worse prognosis in FN-RMS but not in the smaller FP-RMS cohort. CONCLUSION Our study demonstrates that baseline ctDNA detection is feasible and is prognostic in IR RMS.

PURPOSE Monoclonal antibodies directed against insulin-like growth factor-1 receptor (IGF-1R) have shown activity in patients with relapsed Ewing sarcoma. The primary objective of Children's Oncology Group trial AEWS1221 was to determine if the addition of the IGF-1R monoclonal antibody ganitumab to interval-compressed chemotherapy improves event-free survival (EFS) in patients with newly diagnosed metastatic Ewing sarcoma. METHODS Patients were randomly assigned 1:1 at enrollment to standard arm (interval-compressed vincristine/doxorubicin/cyclophosphamide alternating once every 2 weeks with ifosfamide/etoposide = VDC/IE) or to experimental arm (VDC/IE with ganitumab at cycle starts and as monotherapy once every 3 weeks for 6 months after conventional therapy). A planned sample size of 300 patients was projected to provide 81% power to detect an EFS hazard ratio of 0.67 or smaller for the experimental arm compared with the standard arm with a one-sided α of .025. RESULTS Two hundred ninety-eight eligible patients enrolled (148 in standard arm; 150 in experimental arm). The 3-year EFS estimates were 37.4% (95% CI, 29.3 to 45.5) for the standard arm and 39.1% (95% CI, 31.3 to 46.7) for the experimental arm (stratified EFS-event hazard ratio for experimental arm 1.00; 95% CI, 0.76 to 1.33; 1-sided, P = .50). The 3-year overall survival estimates were 59.5% (95% CI, 50.8 to 67.3) for the standard arm and 56.7% (95% CI, 48.3 to 64.2) for the experimental arm. More cases of pneumonitis after radiation involving thoracic fields and nominally higher rates of febrile neutropenia and ALT elevation were reported on the experimental arm. CONCLUSION Ganitumab added to interval-compressed chemotherapy did not significantly reduce the risk of EFS event in patients with newly diagnosed metastatic Ewing sarcoma, with outcomes similar to prior trials without IGF-1R inhibition or interval compression. The addition of ganitumab may be associated with increased toxicity.

Purpose: Treatment options for recurrent or refractory Ewing's sarcoma (ES) are limited. Vigil is a novel autologous tumor cell therapy expressing bi-shRNA furin/GMCSF plasmid, which previously demonstrated monotherapy activity in advanced ES. Herein we report safety and evidence of benefit to Vigil for ES as potential treatment. Patients and Methods: In this pilot trial, eligible patients with recurrent or refractory ES who failed initial standard-of-care therapy received treatment with temozolomide (TEM) 100 mg/m2/day oral and irinotecan (IRI) 50 mg/m2/day oral, Days 1 to 5, in combination with Vigil (1 × 106–107 cells/mL/day intradermal, Day 15), every 21 days (Vigil/TEM/IRI). Objective response rate (ORR) by RECIST v1.1, progression-free survival (PFS), and overall survival (OS) were assessed. Circulating tumor (ct) DNA analysis was done by patient-specific droplet digital PCR on baseline and serially collected on-treatment samples. Results: Eight of 10 enrolled patients were evaluable for safety and efficacy (mean age 24.6; 12.6–46.1 years old); 2 did not receive Vigil. Seven of 8 patients previously received TEM/IRI. No Vigil-related adverse events were reported. Common ≥Grade 3 chemotherapy-related toxicity included neutropenia (50%) and thrombocytopenia (38%). We observed two partial response patients by RECIST; both showed histologic complete response without additional cancer therapy. Median PFS was 8.2 months (95% confidence interval, 4.3–NA). Five patients showed stable disease or better for ≥6 months. Patient-specific EWS/FLI1 ctDNA was detectable in all 8 evaluable patients at baseline. Changes in ctDNA levels corresponded to changes in disease burden. Conclusions: Results demonstrated safety of combination Vigil/TEM/IRI.

Background:Treatment options for recurrent or refractory Ewing's sarcoma (ES) are limited. Vigil is a novel autologous tumor cell therapy expressing bi-shRNA furin/GMCSF plasmid, which previously demonstrated monotherapy activity in advanced ES. Herein we report safety and evidence of benefit to Vigil for ES as potential treatment.Patients and Methods:In this pilot trial, eligible patients with recurrent or refractory ES who failed initial standard-of-care therapy received treatment with temozolomide (TEM) 100 mg/m2/day oral and irinotecan (IRI) 50 mg/m2/day oral, Days 1 to 5, in combination with Vigil (1 × 106–107 cells/mL/day intradermal, Day 15), every 21 days (Vigil/TEM/IRI). Objective response rate (ORR) by RECIST v1.1, progression-free survival (PFS), and overall survival (OS) were assessed. Circulating tumor (ct) DNA analysis was done by patient-specific droplet digital PCR on baseline and serially collected on-treatment samples.Results:Eight of 10 enrolled patients were evaluable for safety and efficacy (mean age 24.6; 12.6–46.1 years old); 2 did not receive Vigil. Seven of 8 patients previously received TEM/IRI. No Vigil-related adverse events were reported. Common ≥Grade 3 chemotherapy-related toxicity included neutropenia (50%) and thrombocytopenia (38%). We observed two partial response patients by RECIST; both showed histologic complete response without additional cancer therapy. Median PFS was 8.2 months (95% confidence interval, 4.3–NA). Five patients showed stable disease or better for ≥6 months. Patient-specific EWS/FLI1 ctDNA was detectable in all 8 evaluable patients at baseline. Changes in ctDNA levels corresponded to changes in disease burden.Conclusions:Results demonstrated safety of combination Vigil/TEM/IRI.

Variants identified from WES of 11 cell lines after filtering.

GSK-LSD1, Doxorubicin and AraC have no effect on KDM1A and KDM1B expression

Protein expression of ER-stress response genes following treatment with SP-2509

This file contains supplementary methods, supplementary references, supplementary figures S1-S6, supplementary tables S1, S3-S6, S8-S15, and S17, and legends for all supplementary figures and tables. Supplementary Figure S1. Summary of Ewing sarcoma tumor and cell line cohorts. Supplementary Figure S2. RNASeq validation of mutations identified by WES and WGS. Supplementary Figure S3. Power calculations for the detection of significantly mutated genes. Supplementary Figure S4. Copy number variants in Ewing sarcoma tumors. Supplementary Figure S5. ETS1 deletions associated with EWS/FLI rearrangements. Supplementary Figure S6. Copy number variants in diagnostic vs. treated Ewing sarcoma tumors. Supplementary Figure S7. SCNAs in paired Ewing sarcoma tumor samples. Supplementary Table S1. Tissue samples. Supplementary Table S3. Multiply mutated genes. Supplementary Table S4. Rearrangements and fusions detected by WGS and RNASeq. Supplementary Table S5. Mutational significance for tumor/normal pairs. Supplementary Table S6. Rate of coding mutations in Ewing sarcoma tumors. Supplementary Table S8. Mutational significance for all tumors. Supplementary Table S9. Gene set enrichment analyses of mutated genes. Supplementary Table S10. Significance of arm-level SCNAs. Supplementary Table S11. Multiply mutated genes of interest in all sample cohorts. Supplementary Table S12. Immunohistochemical staining of STAG2 in Ewing sarcoma tumors. Supplementary Table S13. Gene set enrichment analyses of STAG2 expressing vs. STAG2 loss. Supplementary Table S14. Gene and gene-fragment RPKM Values for EWS and ETS genes. Supplementary Table S15. ETS1 copy-number changes. Supplementary Table S17. Gene set enrichment analyses of tumors vs. cell lines.

Targeted KDM1A inhibition and EWS/FLI knockdown induces similar transcriptional signatures

Hypersensitive SP-2509 cell lines display similar transcriptomic profiles

Cell line source/culture conditions, primer/shRNA sequences, antibodies utilized in this study and additional methods

Validation of KDM1A knockdown in Ewing sarcoma cells

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 –1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902–16. ©2018 AACR.

Pediatric Ewing sarcoma is characterized by the expression of chimeric fusions of EWS and ETS family transcription factors, representing a paradigm for studying cancers driven by transcription factor rearrangements. In this study, we describe the somatic landscape of pediatric Ewing sarcoma. These tumors are among the most genetically normal cancers characterized to date, with only EWS–ETS rearrangements identified in the majority of tumors. STAG2 loss, however, is present in more than 15% of Ewing sarcoma tumors; occurs by point mutation, rearrangement, and likely nongenetic mechanisms; and is associated with disease dissemination. Perhaps the most striking finding is the paucity of mutations in immediately targetable signal transduction pathways, highlighting the need for new therapeutic approaches to target EWS–ETS fusions in this disease.Significance: We performed next-generation sequencing of Ewing sarcoma, a pediatric cancer involving bone, characterized by expression of EWS–ETS fusions. We found remarkably few mutations. However, we discovered that loss of STAG2 expression occurs in 15% of tumors and is associated with metastatic disease, suggesting a potential genetic vulnerability in Ewing sarcoma. Cancer Discov; 4(11); 1326–41. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 1243

Cytotoxic effects of the irreversible KDM1A inhibitors GSK-LSD1 and Tranylcypromine

supplementary table

Serrated polyps are pathological neoplastic lesions in the colon with subtle gross morphology leading to underreporting during colonoscopy. While detection rates are increasing in average-risk adult screening colonoscopy, the rate of detection during pediatric colonoscopy is unknown. Serrated polyposis syndrome is characterized by the presence of multiple serrated polyps in the colon and an increased risk of developing colorectal cancer. Cancer prevention relies on early recognition, endoscopic clearance of all polyps5 mm, and continued interval surveillance or prophylactic colectomy. We report the diagnosis and management of serrated polyposis syndrome in a young adolescent patient and highlight the subtle features of serrated polyps that may go unrecognized leading to underreporting in childhood.

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

Purpose: Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss, and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize patient-derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies. Experimental Design: We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA sequencing, and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling. Results: We show that LMS PDX maintain the histologic appearance, copy-number alterations, and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX cocluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional cyclin-dependent kinase (CDK) inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth. Conclusions: Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for patients with LMS.

Purpose:Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss, and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize patient-derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies.Experimental Design:We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA sequencing, and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling.Results:We show that LMS PDX maintain the histologic appearance, copy-number alterations, and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX cocluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional cyclin-dependent kinase (CDK) inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth.Conclusions:Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for patients with LMS.

Supplementary Table from Preclinical Modeling of Leiomyosarcoma Identifies Susceptibility to Transcriptional CDK Inhibitors through Antagonism of E2F-Driven Oncogenic Gene Expression

Purpose:Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients.Experimental Design:Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines in vitro and in multiple xenograft models of Ewing sarcoma.Results:Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B–selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma.Conclusions:FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.

Supplemental Table S7. Reverse Phase Protein Array (RPPA) data. Total protein and phosphorylation levels in untreated and treated A673 and TC32 cells are normalized to total protein levels in each sample.

Supplementary Figure from Preclinical Modeling of Leiomyosarcoma Identifies Susceptibility to Transcriptional CDK Inhibitors through Antagonism of E2F-Driven Oncogenic Gene Expression

Supplemental Methods and Figures Supplemental Figure S1. Aurora kinase expression in Ewing sarcoma Supplemental Figure S2. Effects of cell growth on response to AZD-1152 as a function of duration of treatment. Supplemental Figure S3. Aurora kinase and FAK inhibitor combinations are synergistic in Ewing sarcoma cell lines Supplemental Figure S4. Response of Ewing cell lines treated with combinations of Aurora kinase and FAK inhibitors Supplemental Figure S5. Cell cycle and apoptotic effects of Aurora kinase B knock out in Ewing sarcoma cell lines Supplemental Figure S6. PYK2 is poorly expressed in Ewing sarcoma cells Supplemental Figure S7. Zebrafish and Murine studies

Supplementary Data from Preclinical Modeling of Leiomyosarcoma Identifies Susceptibility to Transcriptional CDK Inhibitors through Antagonism of E2F-Driven Oncogenic Gene Expression

Purpose:We sought to determine whether the detection of circulating tumor DNA (ctDNA) in samples of patients undergoing chemotherapy for advanced leiomyosarcoma (LMS) is associated with objective response or survival.Experimental Design:Using ultra–low-passage whole-genome sequencing (ULP-WGS) of plasma cell-free DNA from patients treated on a prospective clinical trial, we tested whether detection of ctDNA evaluated prior to the start of therapy and after two cycles of chemotherapy was associated with treatment response and outcome. Associations between detection of ctDNA and pathologic measures of disease burden were evaluated.Results:We found that ctDNA was detectable by ULP-WGS in 49% patients prior to treatment and in 24.6% patients after two cycles of chemotherapy. Detection of pretreatment ctDNA was significantly associated with a lower overall survival [HR, 1.55; 95% confidence interval (CI), 1.03–2.31; P = 0.03] and a significantly lower likelihood of objective response [odds ratio (OR), 0.21; 95% CI, 0.06–0.59; P = 0.005]. After two cycles of chemotherapy, patients who continued to have detectable levels of ctDNA experienced a significantly worse overall survival (HR, 1.77; 95% CI, 1–3.14; P = 0.05) and were unlikely to experience an objective response (OR, 0.05; 95% CI, 0–0.39; P = 0.001).Conclusions:Our results demonstrate that detection of ctDNA is associated with outcome and objective response to chemotherapy in patients with advanced LMS. These results suggest that liquid biopsy assays could be used to inform treatment decisions by recognizing patients who are likely and unlikely to benefit from chemotherapy.See related commentary by Kasper and Wilky, p. 2480

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PDF file - 33K, Sequences for pLKO.1 FAK-targeting and CAS-targeting shRNAs (S1); Median fluorescent intensity for six Ewing sarcoma cell lines and 293FT cells measuring the phosphorylation level of 62 tyrosine kinases (S2); PF-562271 downregulates mTOR, AKT, and S6 phosphorylation (S3).

PDF file - 62K, Treatment of two Ewing sarcoma cell lines with A, dasatinib (SRC inhibitor) for 8 hours or B, PD0325901 (MEK inhibitor) for 72 hours eliminates phosphorylation of SRC and ERK, respectively (S1); Transduction with five FAK-directed shRNAs downregulate FAK expression compared to a control shRNA in the A673 Ewing sarcoma cell line (S2); Treatment of A673 and TC32 Ewing sarcoma cell lines with PF-562271 reduced cell growth (S3); Treatment of Ewing sarcoma cell lines with PF- 562271 impairs cell growth (S4); PYK2 is variably expressed in Ewing sarcoma cell lines (S5).

Limited progress has been made in the treatment of advanced-stage pediatric solid tumors despite the accelerated pace of cancer discovery over the last decade. Tyrosine kinase inhibition is one tractable therapeutic modality for treating human malignancy. However, little is known about the kinases critical to the development or maintenance of many pediatric solid tumors such as Ewing sarcoma. Using a fluorescent, bead-based technology to profile activated tyrosine kinases, we identified focal adhesion kinase (FAK, PTK2) as a candidate target in Ewing sarcoma. FAK is a tyrosine kinase critical for cellular adhesion, growth, and survival. As such, it is a compelling target for cancer-based therapy. In this study, we have shown that FAK is highly phosphorylated in primary Ewing sarcoma tumor samples and that downregulation of FAK by short hairpin RNA and treatment with a FAK-selective kinase inhibitor, PF-562271, impaired growth and colony formation in Ewing sarcoma cell lines. Moreover, treatment of Ewing sarcoma cell lines with PF-562271 induced apoptosis and led to downregulation of AKT/mTOR and CAS activity. Finally, we showed that small-molecule inhibition of FAK attenuated Ewing sarcoma tumor growth in vivo. With FAK inhibitors currently in early-phase clinical trials for adult malignancies, these findings may bear immediate relevance to patients with Ewing sarcoma. Cancer Res; 73(9); 2873–83. ©2013 AACR.

PURPOSE Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS ( P = .04). The most frequently altered genes in the primary cohort were TP 53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort ( P = .015) and the validation cohort ( P = .012). CONCLUSION The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Ewing sarcoma is the second most common pediatric bone cancer in children and young adults. The standard of care therapy consists of chemotherapy, surgery, and radiation. Despite therapeutic advances, metastatic and relapsed Ewing sarcoma have poor outcomes. This is partially because transcription factors, such as the Ewing sarcoma oncoprotein EWS-FLI1, are difficult targets for the development of small molecules. Novel synergistic combination treatments are needed, and drug repurposing efforts can fast track potential therapies into clinical trials. To identify synergistic anti-Ewing combinations of targeted cancer agents, we conducted an initial library screen of 26 tyrosine kinase inhibitors, cell cycle inhibitors, and chemotherapies on two Ewing cell lines. While the screening results revealed multiple synergistic combinations of tyrosine kinase inhibitors and cell cycle inhibitors, we focused our follow-up validation studies on combinations that met at least one of the following criteria: 1) agents that exhibited additivity when combined with VEGFR inhibitors, a class of agents already shown to having clinical efficacy in Ewing sarcoma and 2) combinations involving approved FDA drugs that could be rapidly translated into early phase trials for patients with Ewing sarcoma. Efficacy of selected synergistic combinations were then tested in vitro on a larger collection of Ewing cell lines. Based on these results, two combinations, one with a VEGFR inhibitor and one combination of FDA approved agents, was tested in an aggressive Ewing sarcoma xenograft mouse model for efficacy and impact on survival. We demonstrated that the combination of ribociclib, a CDK 4/6 inhibitor, and regorafenib, a VEGFR inhibitor, exhibited additivity and synergy in Ewing cell lines. Xenografted mice treated with this combination had significantly impaired tumor proliferation in a subcutaneous tumor model and prolonged survival (median 35 days) compared to mice treated with vehicle (median 28 days) or single-agent therapy. Similarly, cell lines treated with copanlisib, a PI3K inhibitor, and ribociclib, two FDA-approved drugs, synergistically inhibited cell line viability in vitro and impaired xenograft proliferation in vivo. This combination also significantly improved survival (median 48 days) in a mouse xenograft compared to treatment with vehicle (median 27 days) or either drug alone. This work demonstrates that CDK 4/6 inhibitors show synergistic activity with both VEGFR inhibitors and PI3K inhibitors in Ewing Sarcoma by impairing the growth of cultured Ewing cells and improving the survival of mice with Ewing sarcoma. We believe that pre-clinical validation of novel approved drug combinations in Ewing sarcoma will accelerate the development of early phase trials for patients with relapsed refractory disease, a group of patients greatly in need of new therapeutic opportunities. Citation Format: Maria Ana Isabel C. De Los Santos, Anne-Florence Blandin, Alejandra E. Aguilar, Matthew Hall, Min Shen, Ya-Qin Zhang, Brian D. Crompton. Identifying novel combination therapies for Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6732.

Context.— Molecular diagnostics play an increasing role in the diagnosis of Ewing sarcoma. The type of molecular testing used in clinical practice has been poorly described. Objective.— To describe patterns of translocation testing for newly diagnosed Ewing sarcoma. Design.— Children's Oncology Group (COG) trial AEWS1221 was a phase III randomized trial enrolling patients with newly diagnosed metastatic Ewing sarcoma from 2014 to 2019. Patients were required to have a histologic diagnosis of Ewing sarcoma, but translocation testing was not required. Sites provided types and results of any molecular diagnostics performed. Results.— Data from 305 enrolled patients were available. The most common type of molecular testing was fluorescence in situ hybridization (FISH) performed on the primary tumor (236 of 305 patients; 77.4%), with positive testing for an EWSR1 or FUS translocation in 211 (89.4%). Reverse transcription–polymerase chain reaction (RT-PCR) on the primary tumor was performed in 61 of 305 patients (20%), with positive results in 48 of 61 patients (78.7%). Next-generation sequencing was reported in 7 patients for the primary tumor and in 3 patients for metastatic sites. For all types of testing on either primary or metastatic tumor, 16 of 305 patients (5.2%) had no reported translocation testing. When evaluating all results from all testing, 44 of 305 patients (14.4%) lacked documentation of an abnormality consistent with a molecular diagnosis of Ewing sarcoma. Conclusions.— COG sites enrolling in a Ewing sarcoma trial have high rates of testing by FISH or PCR. A small proportion of patients have no translocation testing on either primary or metastatic sites. Next-generation sequencing techniques are not yet commonly used in this context.

Background Ewing sarcoma (EWS) is an aggressive sarcoma with no validated molecular biomarkers. We aimed to determine the frequency of STAG2 protein loss by immunohistochemistry (IHC) and whether loss of expression is associated with outcome. Methods We performed a retrospective cohort study of patients with EWS enrolled to Children’s Oncology Group studies. We obtained unstained slides from 235 patients and DNA for sequencing from 75 patients. STAG2 expression was tested for association with clinical features and survival was estimated using Kaplan–Meier methods with log-rank tests. Results In total, 155 cases passed quality control for STAG2 IHC. STAG2 expression in 20/155 cases could not be categorised with the limited available tissue, leaving 135 patients with definitive STAG2 IHC. In localised and metastatic disease, STAG2 was lost in 29/108 and 6/27 cases, respectively. Among patients with IHC and sequencing, 0/17 STAG2 expressing cases had STAG2 mutations, and 2/7 cases with STAG2 loss had STAG2 mutations. Among patients with localised disease, 5-year event-free survival was 54% (95% CI 34–70%) and 75% (95% CI 63–84%) for patients with STAG2 loss vs. expression (P = 0.0034). Conclusion STAG2 loss of expression is identified in a population of patients without identifiable STAG2 mutations and carries a poor prognosis.

PURPOSE The utility of circulating tumor DNA (ctDNA) analyses has not been established in the risk stratification of Wilms tumor (WT). We evaluated the detection of ctDNA and selected risk markers in the serum and urine of patients with WT and compared findings with those of matched diagnostic tumor samples. PATIENTS AND METHODS Fifty of 395 children with stage III or IV WT enrolled on Children's Oncology Group trial AREN0533 had banked pretreatment serum, urine, and tumor available. Next-generation sequencing was used to detect ctDNA. Copy-number changes in 1q, 16q, and 1p, and single-nucleotide variants in serum and urine were compared with tumor biopsy data. Event-free survival (EFS) was compared between patients with and without ctDNA detection. RESULTS ctDNA was detected in the serum of 41/50 (82%) and in the urine in 13/50 (26%) patients. Agreement between serum ctDNA detection and tumor sequencing results was as follows: 77% for 1q gain, 88% for 16q deletions, and 70% for 1p deletions, with ĸ-coefficients of 0.56, 0.74, and 0.29, respectively. Sequencing also demonstrated that single-nucleotide variants detected in tumors could be identified in the ctDNA. There was a trend toward worse EFS in patients with ctDNA detected in the serum (4-year EFS 80% v 100%, P = .14). CONCLUSION ctDNA demonstrates promise as an easily accessible prognostic biomarker with potential to detect tumor heterogeneity. The observed trend toward more favorable outcome in patients with undetectable ctDNA requires validation. ctDNA profiling should be further explored as a noninvasive diagnostic and prognostic tool in the risk-adapted treatment of patients with WT.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies in a systematic manner remains challenging in part due to the lack of faithful preclinical in vitro models. We established ten short-term patient-derived WT cell lines and characterized these models using low-coverage whole genome sequencing, whole exome sequencing and RNA-sequencing, which demonstrated that these ex-vivo models faithfully recapitulate WT biology. We then performed targeted RNAi and CRISPR-Cas9 loss-of-function screens and identified the nuclear export genes (XPO1 and KPNB1) as strong vulnerabilities. We observed that these models are sensitive to nuclear export inhibition using the FDA approved therapeutic agent, selinexor (KPT-330). Selinexor treatment of FHWT suppressed TRIP13 expression, which was required for survival. We further identified in vitro and in vivo synergy between selinexor and doxorubicin, a chemotherapy used in high risk FHWT. Taken together, we identified XPO1 inhibition with selinexor as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

More knowledge is needed around the role and importance of specific genes in germline predisposition to Ewing sarcoma to inform biological investigation and clinical practice. In this study, we evaluated the enrichment of pathogenic germline variants in Ewing sarcoma relative to other pediatric sarcoma subtypes, as well as patterns of inheritance of these variants. We carried out an ancestry-matched case-control analysis to screen for enrichment of pathogenic germline variants in 141 established cancer predisposition genes in 1138 individuals with pediatric sarcoma diagnoses (222 Ewing sarcoma cases) relative to identically processed cancer-free controls. Findings in Ewing sarcoma were validated with an additional cohort of 425 individuals, and 301 Ewing sarcoma parent-proband trios were analyzed for inheritance patterns of identified pathogenic variants. A distinct pattern of pathogenic germline variants was seen in Ewing sarcoma relative to other sarcoma subtypes.FANCCwas the only gene with an enrichment signal for heterozygous pathogenic variants in the discovery Ewing sarcoma cohort (OR 14.4, 95% CI 3.5 – 51.2, p = 0.002, FDR = 0.28). This enrichment inFANCCheterozygous pathogenic variants was seen again in the Ewing sarcoma validation cohort (OR 5.1, 95% CI 1.2 – 18.5, p = 0.03, single hypothesis), representing a broader importance of genes involved in DNA damage repair, which were also nominally enriched in Ewing sarcoma cases. Pathogenic variants in DNA damage repair genes were acquired through autosomal inheritance. Our study provides new insight into germline risk factors contributing to Ewing sarcoma pathogenesis.

More knowledge is needed regarding germline predisposition to Ewing sarcoma to inform biological investigation and clinical practice. Here, we evaluated the enrichment of pathogenic germline variants in Ewing sarcoma relative to other pediatric sarcoma subtypes, as well as patterns of inheritance of these variants. We carried out European-focused and pan-ancestry case-control analyses to screen for enrichment of pathogenic germline variants in 141 established cancer predisposition genes in 1,147 individuals with pediatric sarcoma diagnoses (226 Ewing sarcoma, 438 osteosarcoma, 180 rhabdomyosarcoma, and 303 other sarcoma) relative to identically processed cancer-free control individuals. Findings in Ewing sarcoma were validated with an additional cohort of 430 individuals, and a subset of 301 Ewing sarcoma parent-proband trios was analyzed for inheritance patterns of identified pathogenic variants. A distinct pattern of pathogenic germline variants was seen in Ewing sarcoma relative to other sarcoma subtypes. FANCC was the only gene with an enrichment signal for heterozygous pathogenic variants in the European Ewing sarcoma discovery cohort (three individuals, OR 12.6, 95% CI 3.0-43.2, p = 0.003, FDR = 0.40). This enrichment in FANCC heterozygous pathogenic variants was again observed in the European Ewing sarcoma validation cohort (three individuals, OR 7.0, 95% CI 1.7-23.6, p = 0.014), representing a broader importance of genes involved in DNA damage repair, which were also nominally enriched in individuals with Ewing sarcoma. Pathogenic variants in DNA damage repair genes were acquired through autosomal inheritance. Our study provides new insight into germline risk factors contributing to Ewing sarcoma pathogenesis.

PURPOSE Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks. MATERIALS AND METHODS Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation. RESULTS One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events. CONCLUSION A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.

Purpose: We sought to determine whether the detection of circulating tumor DNA (ctDNA) in samples of patients undergoing chemotherapy for advanced leiomyosarcoma (LMS) is associated with objective response or survival. Experimental Design: Using ultra–low-passage whole-genome sequencing (ULP-WGS) of plasma cell-free DNA from patients treated on a prospective clinical trial, we tested whether detection of ctDNA evaluated prior to the start of therapy and after two cycles of chemotherapy was associated with treatment response and outcome. Associations between detection of ctDNA and pathologic measures of disease burden were evaluated. Results: We found that ctDNA was detectable by ULP-WGS in 49% patients prior to treatment and in 24.6% patients after two cycles of chemotherapy. Detection of pretreatment ctDNA was significantly associated with a lower overall survival [HR, 1.55; 95% confidence interval (CI), 1.03–2.31; P = 0.03] and a significantly lower likelihood of objective response [odds ratio (OR), 0.21; 95% CI, 0.06–0.59; P = 0.005]. After two cycles of chemotherapy, patients who continued to have detectable levels of ctDNA experienced a significantly worse overall survival (HR, 1.77; 95% CI, 1–3.14; P = 0.05) and were unlikely to experience an objective response (OR, 0.05; 95% CI, 0–0.39; P = 0.001). Conclusions: Our results demonstrate that detection of ctDNA is associated with outcome and objective response to chemotherapy in patients with advanced LMS. These results suggest that liquid biopsy assays could be used to inform treatment decisions by recognizing patients who are likely and unlikely to benefit from chemotherapy. See related commentary by Kasper and Wilky, p. 2480

Somatic gene translocations are key to making an accurate diagnosis in many cancers including many pediatric sarcomas. Currently available molecular diagnostic approaches to identifying somatic pathognomonic translocations have limitations such as minimal multiplexing, high cost, complex computational requirements, or slow turnaround times. We sought to develop a new fusion-detection assay optimized to mitigate these challenges. To accomplish this goal, we developed a highly sensitive multiplexed digital PCR-based approach that can identify the gene partners of multiple somatic fusion transcripts. This assay was validated for specificity with cell lines and synthetized DNA fragments. Assay sensitivity was optimized using a tiered amplification approach for fusion detection from low input and/or degraded RNA. The assay was then tested for the potential application of fusion detection from FFPE tissue and liquid biopsy samples. We found that this multiplexed PCR approach was able to accurately identify the presence of seven different targeted fusion transcripts with a turnaround time of 1 to 2 days. The addition of a tiered amplification step allowed the detection of targeted fusions from as little as 1 pg of RNA input. We also identified fusions from as little as two unstained slides of FFPE tumor biopsy tissue, from circulating tumor cells collected from tumor-bearing mice, and from liquid biopsy samples from patients with known fusion-positive cancers. We also demonstrated that the assay could be easily adapted for additional fusion targets. In summary, this novel assay detects multiple somatic fusion partners in biologic samples with low tumor content and low-quality RNA in less than two days. The assay is inexpensive and could be applied to surgical and liquid biopsies, particularly in places with inadequate resources for more expensive and expertise-dependent assays such as next-generation sequencing.

Patients who are diagnosed with osteosarcoma (OS) today receive the same therapy that patients have received over the last 4 decades. Extensive efforts to identify more effective or less toxic regimens have proved disappointing. As we enter a postgenomic era in which we now recognize OS not as a cancer of mutations but as one defined by p53 loss, chromosomal complexity, copy number alteration, and profound heterogeneity, emerging threads of discovery leave many hopeful that an improving understanding of biology will drive discoveries that improve clinical care. Under the organization of the Bone Tumor Biology Committee of the Children's Oncology Group, a team of clinicians and scientists sought to define the state of the science and to identify questions that, if answered, have the greatest potential to drive fundamental clinical advances. Having discussed these questions in a series of meetings, each led by invited experts, we distilled these conversations into a series of seven Provocative Questions. These include questions about the molecular events that trigger oncogenesis, the genomic and epigenomic drivers of disease, the biology of lung metastasis, research models that best predict clinical outcomes, and processes for translating findings into clinical trials. Here, we briefly present each Provocative Question, review the current scientific evidence, note the immediate opportunities, and speculate on the impact that answered questions might have on the field. We do so with an intent to provide a framework around which investigators can build programs and collaborations to tackle the hardest problems and to establish research priorities for those developing policies and providing funding.

Background: Recapitulation of the full spectrum of genomic changes driving patient tumors have resulted in increased use of patient-derived xenograft (PDX) models in studies of basic cancer biology and preclinical drug development. Given the translational potential of PDXs and limited availability of pediatric cancer models, we established a PDX program to expand the existing collection of pediatric PDXs in the community and enable pre- and post-clinical studies. Methods: PDX generation requests were integrated into clinical workflows to maximize identification of eligible patients for informed consent and tissue collection at Memorial Sloan Kettering Cancer Center. Methodologies for tissue procurement and cryopreservation were optimized to facilitate implantation into host immunodeficient mice and enable multi-institutional tissue exchange for model building. A bioinformatics pipeline was established to allow molecular validation of engrafted PDXs using a next-generation targeted gene panel (MSK-IMPACT) evaluating concordance based on acquired mutations, copy number alterations and clonal structure. Results: Between November 2016 - October 2021, 379 PDX models were developed (265 distinct models) representing 69 discrete diagnoses. Sarcoma represents the most common model type (50 discrete osteosarcoma, 20 desmoplastic small round cell tumor, 14 Ewing sarcoma, 24 rhabdomyosarcoma, 2 CIC/DUX4 and 2 BCOR-rearranged sarcoma) followed by neuroblastoma (n=35), leukemia (n=44), and Wilms tumor (n=15). While the majority of PDXs were established from recurrent or metastatic tissue, 7 paired diagnostic/pre-therapy and post-therapy or relapse models were generated. Genomic characterization of PDXs demonstrate excellent concordance and recapitulation of single nucleotide variants (90%), structural (88%) and copy number variants (94%) between patient tumor and matched PDX. Discrepancies between matched patient/PDX pairs are due to sub-clonal heterogeneity in source tumors with clonal outgrowth in the PDX. Analysis of serial PDX passages also demonstrate stable recapitulation of the genomic profile. Establishment of a diverse PDX collection allowed preclinical evaluation of 10 targeted agents across a spectrum of pediatric tumors and provided the preclinical rationale for 3 investigator-initiated pediatric clinical trials. Conclusions: Investment in the development of a phenotypically diverse and biologically faithful collection of pediatric PDX models enables the goals of precision medicine. Optimization of PDX workflows and methods has also enabled the development of a pediatric PDX consortium (PROXC - Pediatric Research in Oncology Xenografting Consortium) to further support the development of pre- and post-clinical studies for pediatric cancer. Citation Format: Filemon S. Dela Cruz, Joseph G. McCarter, Daoqi You, Nancy Bouvier, Xinyi Wang, Kristina C. Guillan, Armaan H. Siddiquee, Katie B. Souto, Hongyan Li, Teng Gao, Dominik Glodzik, Daniel Diolaiti, Neerav N. Shukla, Joachim Silber, Umeshkumar K. Bhanot, Faruk Erdem Kombak, Diego F. Coutinho, Shanita Li, Juan E. Arango Ossa, Juan S. Medina-Martinez, Michael V. Ortiz, Emily K. Slotkin, Michael D. Kinnaman, Sameer F. Sait, Tara J. O'Donohue, Marissa Mattar, Maximiliano Meneses, Michael P. LaQuaglia, Todd E. Heaton, Justin T. Gerstle, Nicola Fabbri, Chelsey M. Burke, Irene M. Rodriquez-Sanchez, Christine A. Iacobuzio-Donahue, Julia L. Glade Bender, Ryan D. Roberts, Jason T. Yustein, Nino C. Rainusso, Brian D. Crompton, Elizabeth Stewart, Alejandro Sweet-Cordero, Leanne C. Sayles, Andrika D. Thomas, Michael H. Roehrl, Elisa de Stanchina, Elli Papaemmanuil, Andrew L. Kung. Development of a patient-derived xenograft (PDX) modeling program to enable pediatric precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 704.