Ewing Sarcoma Patients Research Articles

Abstract 3980: Targeting Ewing sarcoma based on RNA expression

Abstract Purpose: Ewing sarcoma (ES) is the second most common malignant bone sarcoma in children and young adults. Current treatment for Ewing sarcoma includes a combination of cytotoxic chemotherapeutic agents and local control with radiation and/or surgery. The survival rate of those with Ewing sarcoma has seen very little improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. The purpose of this study is to identify novel agents currently available for use in Ewing sarcoma. Methods: Novel targeted approaches for the treatment of ES were identified using RNA sequencing performed on 15 ES patient derived cell lines with bioinformatic analysis of gene expression. Targeted agents were tested on patient cell lines at escalating doses. At 48 hours cell viability was assessed with CellTiter-Glo 2.0 and analyzed with GraphPad Prism to determine the IC50. At 24- and 48-hours western blot analysis was performed to evaluate the effect on cell cycle and apoptosis. Combination studies with standard chemotherapy were performed to evaluate for synergy using SynergyFinder 2.0. Results: RNA analysis identified HDAC2, ALK and JAK1 as a potential therapeutic targets. The pan-HDAC inhibitor, Panobinostat, was cytotoxic to all Ewing sarcoma cell lines tested with clinically relevant IC50s (≤200nM) and repressed expression of Cyclin D1 and pRB. ALK inhibition was cytotoxic at doses slightly higher than that of clinical relevance. HDAC and ALK expression levels correlated to cell sensitivity. JAK1 inhibition does not appear to have efficacy at clinically relevant dosing. Panobinostat synergizes with Doxorubicin at clinically relevant doses. Conclusion: This work highlights that RNA expression may identify potential targets which could be effective in treating Ewing sarcoma or potentially improve the efficacy of standard of care drugs. HDAC and ALK inhibition show a decrease in cell viability in ES cell line models. Future studies of HDAC and ALK inhibitors will aim to determine efficacy in xenograft models. Citation Format: Kaitlyn Smith, Divya Gandra, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Giselle S. Sholler, Javier Oesterheld, Erin Trovillion. Targeting Ewing sarcoma based on RNA expression [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 3980.

Abstract 4015: Primary sites affecting the prognosis of Ewing's sarcoma

Abstract Introduction Ewing’s sarcoma (ES) is a small round blue cell malignancy arising from the bone or soft tissue, and most commonly impacts adolescents and young adults. It is difficult to identify those who will likely relapse, however, ES has a very high recurrence rate. Given these realities, the objective of this study was to determine factors influencing the prognosis of ES patients. Methods Clinical and genomic data of 99 patients with ES, were obtained from the cBioPortal. Tumor mutational burden (TMB) and patient survival information were also obtained from the cBioPortal. Results In the data examined, ES had multiple primary sites of disease, including 16 femur, 10 hipbone, 10 rib, 10 tibia, 6 ischium pubis, 5 scapula, 4 fibula, 3 dorsal vertebra, 3 intra-abdominal, 3 lumbar vertebra, and 3 skull. A comparison of survival relationships between different primary sites of ES indicated that overall survival (OS) was significantly different between femur and dorsal vertebra (p = 0.023) and intra-abdominal and hipbone (p = 0.049). However, no significant difference was determined for TMB between the different primary sites. The median value of TMB was 0.23. Data were divided into two groups: high TMB (TMB-H,≥ 0.23 muts/Mb) and low TMB (TMB-L, < 0.23muts/Mb). Interestingly, the OS of patients with TMB-L was significantly higher than that of patients with TMB-H (p = 0.049; 59.45 versus 42.00 months). The presence of metastases at the time of diagnosis was associated with poor OS ( p = 0.023;) and was a clinical factor associated with outcomes having statistical significance. Conclusions Given the heterogeneity in outcomes for patients with ES, additional prognostic factors are needed to optimize clinical decision making. Prognosis was different depending on primary site of ES. Patients with ES in hipbone displayed a better prognosis compared to that in abdomen. Similarly, patients with ES in dorsal vertebra had a significantly higher OS than that in femur. As such, the primary site should perhaps be considered during ES treatment, although there was no significant difference between TMB and primary site. Our results indicate that high TMB is associated with inferior survival for ES and may provide insights for clinical practice. Citation Format: Jun-Qing Mao, Dan Zhao. Primary sites affecting the prognosis of Ewing's sarcoma [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 4015.

Abstract 3147: Natural killer cells mediate tumor-inhibitory functions of USP6 in Ewing sarcoma

Abstract INTRODUCTION: Understanding how Natural Killer (NK) cell activity is regulated by cancer cells is critical for developing NK-cell based immunotherapies. Our studies show that the deubiquitinating enzyme ubiquitin-specific protease-6 (USP6) can induce an immune-stimulatory hot tumor microenvironment in Ewing sarcoma (ES), the 2nd most common pediatric bone cancer. High USP6 expression in ES is associated with significantly improved overall and progression-free survival. We found that USP6 expression in ES cells inhibits xenograft growth in nude mice, but not NSG mice, implicating a role for the innate immune system, particularly cytotoxic NK cells. Notably, USP6 expression enhanced intra-tumoral abundance, activation, and proliferative capacity of NK cells. In vitro cytotoxicity assays confirm that USP6 can directly augment the ability of ES cells to activate NK cells. Based on these observations, we hypothesize that NK cells are essential for USP6-mediated tumor suppression and seek to determine the mechanism by which their cytolytic function is activated by USP6. METHOD: We used ES cell lines A673 and RD-ES expressing USP6 in a doxycycline (dox)-inducible manner, the immortalized human NK cell line NK-92, and primary human and mouse NK cells. Athymic nude or RAG2-/- mice were xenografted subcutaneously with ES cell lines and fed dox-infused water to induce USP6. Immune cell abundance and activation were monitored both in tumors and peripheral blood by flow cytometry. For in vivo depletion of NK cells, 300ug of anti-NK1.1 (PK136) ab was injected i.p. into RAG2-/- mice one day prior to ES xenografting, then once weekly thereafter. RESULTS: USP6 induces in ES cells the surface expression of multiple NK activating ligands, as well as receptors for the apoptotic factor TRAIL (DR5) and Type I/II interferons (IFNAR1 and IFNGR1), both in vitro and in vivo. This leads to enhanced NK activation and renders ES cells hypersensitive to NK-mediated cytotoxicity. IFNy released by activated NK cells feeds back on the USP6-expressing ES cells, inducing synergistic expression of multiple IFNy-inducible genes, including chemokines CXCL9/10 (chemoattractants for NK cells) and ICAM-1 (essential for immunological synapse formation between NK cells and their targets). A potent feed forward loop for NK activation/ES cell killing is thus generated. Concordantly, in RAG2-/- mice, USP6 induces increased intratumoral infiltration and activation of NK cells, and also, strikingly, systemic activation of NK cells. Depletion of NK cells using anti-NK1.1(PK136) completely abrogates USP6-mediated inhibition of ES xenograft growth. CONCLUSION: NK cells are essential for mediating the tumor inhibitory functions of USP6 in ES. We posit that USP6’s ability to activate NK cells both intratumorally and systemically underlies its association with improved ES patient prognosis, which we seek to exploit for therapeutic benefit in future studies. Citation Format: Kanika Jain, Ian C. Henrich, Laura Nicole Quick, Robert A. Young, Margaret M. Chou. Natural killer cells mediate tumor-inhibitory functions of USP6 in Ewing sarcoma [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 3147.

Surgical subtypes and survival in Ewing sarcoma: A National Cancer Database (NCDB) analysis.

e23511 Background: Ewing sarcoma (EWS) is the one of the most common primary malignancies of bone, more commonly affecting the pediatric population Standard of care for EWS currently involves surgical intervention in combination with chemotherapy, in addition to the potential use of preoperative and/or postoperative radiation on a case by case basis. To date, there is not much in the literature reported regarding different surgical subtypes in these patients. Thus, this study aims to analyze survival outcomes amongst the different surgical subtypes in EWS patients. Methods: Using the NCDB, patients diagnosed with EWS between 2004-2018 were identified using ICD-O-3 histology code 9260. The cohort was analyzed to determine if they received any surgical intervention and, if so, which surgical subtype was used. Univariate analysis was then performed assessing patient length of survival for each surgical subtype. Data was analyzed using SPSS and statistical significance was set at α = 0.05. Results: A total of 2208 patients were included that had survival data and had received surgical intervention. The mean survival time of all patients was 118.1 months. Surgical patients survived an average of 137.2 months while nonsurgical patients survived 96.0 months on average (p < .00001). Analysis of surgical subtypes revealed significantly longer survival in patients who underwent radical excision with limb salvage compared to patients who underwent partial resection (p < .05). Patients who received a partial limb amputation survived significantly longer than patients who received a total limb amputation (p < .01). Patients who underwent local tumor excision or radial excision with limb salvage also survived significantly longer than patients with total limb amputation (p < .05 and p < .01, respectively). Conclusions: This study found that receipt of surgery is associated with longer survival in Ewing sarcoma patients. Further, different surgical interventions may influence survival in Ewing sarcoma as patients receiving radical excision with limb salvage fared better than partial resection and partial limb amputation better than total limb amputation. Future studies would benefit from investigating additional variables that may influence receipt of surgical interventions including patient demographics, socioeconomic status, and surgical center type.[Table: see text]

Facility type and it's impact on survival in Ewing's sarcoma: An NCDB analysis.

e23505 Background: Previous studies have reported a significant association between facility type and survival in cancer patients. This study aimed to investigate the impact of treatment facility type on overall survival for Ewing’s Sarcoma. A secondary objective was to identify differences in distribution of demographic variables between facility types. Methods: The National Cancer Database (NCDB) was used to identify patients diagnosed with Ewing’s sarcoma from 2004 to 2018. Four facility types were identified: Academic/Research Programs, Community Cancer Programs, Comprehensive Community Center Programs, and Integrated Network Cancer Programs. Descriptive statistics and Kaplan Meier curves were used to measure survival in these patients and Chi-square was used to evaluate differences in demographics. Data was analyzed using SPSS and statistical significance was set at α = 0.05. Results: Of 269 patients queried, 49% were treated at an academic facility. Academic Programs had the longest median survival of 54 months; Community Cancer Programs had the shortest median survival of 9 months. Statistically significant survival differences were found between all facility types on log-rank comparison (p < 0.050), except for survival between Integrated Network and Community Cancer Programs (p = 0.162). Academic Programs had the highest overall survival when compared to Community Cancer Programs (p = 0.000), Comprehensive Community Cancer Programs (p = 0.026), and Integrated Cancer Networks p = (0.003). Chi-Square analysis revealed an association between facility type and cancer stage at diagnosis (p = 0.044) as well as between facility type and primary payer status (p = 0.026). Notably, 58% of patients treated at Community Programs presented with stage IV disease and 41% were insured by Medicare. Additional log-rank comparisons and Kaplan Meier curves showed private insurance was associated with significantly higher survival compared with Medicaid (p = 0.043), Medicare (p = 0.001) and uninsured patients (p = 0.010). Patients treated at Comprehensive Community (70%) and Academic Programs (67%) had the highest proportion of patients with private insurance. Conclusions: This study showed that Ewing Sarcoma patients treated in an Academic/Research program facility experienced increased survival compared with other facility types. Patients treated at Community Cancer Programs were more likely to present with stage IV disease and were more likely to be on Medicare. Future studies may investigate additional factors that influence potential barriers to care for Ewing Sarcoma patients.[Table: see text]

Genomic co-genotype classification and clinical prognosis in Ewing sarcoma.

e23532 Background: Ewing sarcoma is a highly aggressive round cell mesenchymal tumor that commonly occurs in children and young adults. It is molecularly characterized by chromosomal translocations, most commonly t (11;22), resulting in the abnormal EWSR1-FLI1 fusion. In addition to EWSR1-FLI1, patients may have other genomic variants that can be used for risk stratification. In the present study, we retrospectively investigated the association between co-variant genes and prognosis in patients with Ewing sarcoma. Methods: From 2019 to 2021, 10 patients with Ewing sarcoma were enrolled in the study, and their standard first-line treatment consisted of five-drug regimen chemotherapy including vincristine, doxorubicin (Adriamycin) and cyclophosphamide, alternating with ifosfamide and etoposide (VAC/IE). We carried out whole-exome sequencing on collected tumor samples and matched leukocyte DNA from these patients. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed. Results: In general, median progression-free survival (PFS) of the 10 confirmed Ewing sarcoma (with EWSR1-FLI1 fusion) patients was 10.5 months. We found that 60%, 30%, and 30% of patients had genetic variants (including mutation and copy number variation) in the three signaling pathways of cell cycle, WNT, and NOTCH, respectively. Their PFS was shorter than patients without these variants (log-rank test, p = 0.0345, 0.0053, and 0.0053, respectively). Median PFS was 7 vs 17 months in cell cycle pathway (the former with variants and the latter without variants), 5 vs 14 months in WNT pathway, and 5 vs 14 months in NOTCH pathway. However, there was no difference in PFS between patients with and without variants in other pathways (including TP53, RTK-RAS, MYC, DDR, PI3K) (log-rank test, p＞0.05). Conclusions: We found that the variants in cell cycle, WNT and NOTCH signaling pathway were associated with poor prognosis of Ewing sarcoma. This study provides important molecular markers for risk stratification in patients with Ewing sarcoma, which may provide insights into later clinical trials.

Variations in Ewing sarcoma and rhabdomyosarcoma patient outcomes based on treatment center volume.

e18530 Background: Due to their rarity and requirement for multi-modality care, evidence suggests (and guidelines recommend) patients (pts) with Ewing Sarcoma (ES) and rhabdomyosarcoma (RMS) treated at high volume centres (HVC) with multidisciplinary tumour board discussions (MTDs) have improved outcomes. However, access to care at HVC is challenging for pts who live remotely, such as many pts in British Columbia (BC), Canada. Our study explores the difference in outcomes of patients with ES and RMS based on location of diagnosis and treatment in BC. Methods: A multi-center retrospective chart review of adults diagnosed with ES and RMS Jan 1st 2000 to Dec 31st 2020 was performed for pts undergoing curative intent therapy at one of six provincial cancer centers. All pts underwent expert pathology review. Descriptive statistics were performed. Fisher’s exact and chi-squared tests were used to evaluate associations. Cox proportional hazards model was used to assess for predictors of better survival outcomes by treatment centre. HVCs was defined as those that saw ≥15 patients. Results: 77 patients were included in this analysis; 46 (60%) and 31 (40%) were treated at HVC and LVC respectively. By HVC and LVC respectively, mean age was 36 and 35, 14 (45%) and 20 (43%) were female, 23 (74%) and 26 (57%) had ES, and 11 (35%) and nine (20%) had localized disease. Pts treated at HVCs were more likely to: receive curative intent radiation (p = 0.006), be presented at MTDs (p = 0.042), and less likely to experience disease progression on fist line therapy (p = 0.04) (Table). Time from biopsy to start of chemotherapy (chemo) was 13 days sooner at HVCs vs LVCs (34 vs 47 days, p = 0.12). Initial chemo regimen (p = 0.25) and surgical treatment (p = 0.75) were not significantly different between HVCs vs LVCs. Age, ECOG, stage, and Charlson Comorbidities Index (CCI) were all significant contributors to overall survival (OS). There was no statistically significant difference in OS by treatment center (HR 0.68, 95% CI 0.36 – 1.29). Conclusions: Variations in care exist for both treatment and outcomes amongst ES and RMS pts treated at HVC vs LVC in BC. Pts treated at HVCs were more likely to be presented at MTDs, to receive curative intent radiation, and less likely to experience disease progression. This may reflect differences in access to resources, sarcoma-specific clinician specialists, and familiarity with upfront aggressive treatment protocols. This study can be used to better inform decisions regarding triaging and centralization of curable ES and RMS patients.[Table: see text]

Efficacy of replacing actinomycin-D with carboplatin in Ewing sarcoma consolidation treatment: Single-center experience

BackgroundTreatment of Ewing sarcoma (ES) requires multidisciplinary approach and deficiencies in treatment adversely affect the results. This study included patients diagnosed with ES and aimed to determine the factors affecting prognosis and investigate the efficacy of replacing actinomycin-D with carboplatin in consolidation treatment. MethodsEighty-two pediatric ES patients diagnosed at a single institution between 2005 and 2020 were retrospectively evaluated. Clinical and epidemiological features, treatment modalities, prognostic criteria, and overall survival (OS) rates of patients revieved. In consolidation treatment, 22 patients were treated with actinomycin-D and 32 patients with carboplatin (500 mg/m2/dose), 24 patients could not receive consolidation treatment. The 5- and 10-year OS rates of the patients were compared. ResultsThe 5- and 10-year OS rates of the 82 patients with ES were 46% and 40%, respectively. The 5-year OS rates in the group with localized disease (n = 55) and metastasis (n = 27) at diagnosis were 54% and 26%, respectively (p = 0.006). When evaluated according to the consolidation treatment administered both the 5- and 10-year OS rates of the patients receiving actinomycin-D were 50%. The 5-year OS rate was 58% in the carboplatin group, and the 5- and 10-year OS rates of patients that did not receive consolidation treatment was 20%. ConclusionsSurvival was significantly worse in the group that did not receive consolidation treatment. Furthermore, our results suggested that carboplatin could be used effectively as an alternative to actinomycin-D in ES consolidation treatment.

A novel nomogram and risk classification system predicting the Ewing sarcoma: a population-based study

Ewing sarcoma (ES) is a rare disease that lacks a prognostic prediction model. This study aims to develop a nomogram and risk classification system for estimating the probability of overall survival (OS) of patients with ES. The clinicopathological data of ES were collected from the Surveillance, Epidemiology and Final Results (SEER) database from 2010 to 2018. The primary cohort was randomly assigned to the training set and the validation set. Univariate and multiple Cox proportional hazard analyses based on the training set were performed to identify independent prognostic factors. A nomogram was established to generate individualized predictions of 3- and 5-year OS and evaluated by the concordance index (C-index), the receiver operating characteristic curve (ROC), the calibration curve, the integrated discrimination improvement (IDI) and the net reclassification improvement (NRI). Based on the scores calculated with the nomogram, ES patients were divided into three risk groups to predict their survival. A total of 935 patients were identified, and a nomogram consisting of 6 variables was established. The model provided better C-indices of OS (0.788). The validity of the Cox model assumptions was evaluated through the Schönfeld test and deviance residual. The ROC, calibration curve, IDI and NRI indicated that the nomogram exhibited good performance. A risk classification system was built to classify the risk group of ES patients. The nomogram compares favourably and accurately to the traditional SEER tumour staging systems, and risk stratification provides a more convenient and effective tool for clinicians to optimize treatment options.

Long-term outcome and relapse patterns in Ewing sarcoma patients with extensive lung/pleural metastases after a complete response to systemic therapy.

BackgroundEwing sarcoma (ES) is sensitive to systemic therapy, including chemotherapy and anti-angiogenesis Tyrosine Kinase Inhibitors(aaTKIs). However, the prognosis of patients with metastatic disease remains poor. Recurrence or distant metastasis after a complete response (CR) or near-CR due to systemic therapy is not rare.MethodsWe reviewed data from 187 ES patients between 2014–2019 treated at a single institute in China. Patients with extensive lung/pleural metastases (L/Pmeta) who had a CR or near-CR after first- or second-line chemotherapy with or without aaTKIs were retrospectively enrolled. Event-free survival (EFS) and overall survival (OS) were determined using the Kaplan–Meier method. For patients who had L/P recurrence, images were reviewed to define the exact location of each recurrent lesion, compared with the primary L/P lesion before chemotherapy and summarized as the relapse pattern.ResultsSeventeen patients and 21 cases of CR/nCR (5 by VDC/IE, 3 by VIT, and 13 by AVI) were finally analyzed. Median follow-up for surviving patients was 39.6 (range, 14.5–60.9) months. Median EFS and OS were 9.3 (95% confidence interval [CI], 2.0–16.6) months and 37.5 (95% CI, 21.8–53.1) months, respectively. The 2-year EFS was 19% and the 2-year OS was 70.6%, respectively. Most patients (82.4%) received whole lung irradiation (WLI). Lung/pleural relapse occurred in 71.4% (15/21) of CR/nCR cases. Most notably, all recurrent lesions exactly coincided with the original metastatic lesions before chemotherapy (exactly in situ) in 9 of the 15 recurrent cases, which was thus the major relapse pattern, whereas 42.9% had distant metastases other than L/Pmeta.ConclusionsSurvival of ES patients with extensive L/Pmeta remains poor, even if they have a CR after systemic therapy. Recurrence exactly in situ is the major relapse pattern. WLI is not sufficient to prevent local recurrence in lung or pleura. More aggressive local treatment for metastatic lesions is warranted.

New Approaches Promise to Improve Local Ewing Sarcoma Results.

The study by Whelan and colleagues showed that addition of busulfan and melphalan conditioning and autologous stem cell rescue to conventional EURO-E.W.I.N.G STUDY chemotherapy in local nonmetastatic Ewing sarcoma improves prognosis. However, almost 30% of these study patients will have relapsed before this stage of therapy is reached, and 78% of his patients were at high risk because of inadequate response to the initial chemotherapy given. Further improvement could be achieved by the integration of other novel advances with this approach. Ash and colleagues have shown that the separation of such cases into high- and low-risk groups by using CD56 negativity of the tumor cells is an improvement over current methods with a 100% 10-year progression-free survival in CD56- nonpelvic local isolated Ewing sarcoma patients. Their patients were treated on the SCMCIE 94 protocol, associated with no relapses before 30 months in 24 consecutive patients independent of the CD status. Integration of these novel approaches in diagnosis and treatment would allow truly high-risk patients, who would benefit from the procedure, to reach the busulfan and melphalan stage of therapy and delineate those patients who can be cured without such therapy. Details of the SCMCIE 94 protocol are given and the possible reasons for the different relapse patterns are discussed.

A Visualized Dynamic Prediction Model for Lymphatic Metastasis in Ewing's Sarcoma for Smart Medical Services.

BackgroundThis study aims to predict the lymphatic metastasis in Ewing's sarcoma (ES) patients by nomogram. The risk of lymphatic metastasis in patients with ES was predicted by the built model, which provided guidance for the clinical diagnosis and treatment planning.MethodsA total of 929 patients diagnosed with ES were enrolled from the year of 2010 to 2016 in the Surveillance, Epidemiology, and End Results (SEER) database. The nomogram was established to determine predictive factors of lymphatic metastasis according to univariate and multivariate logistic regression analysis. The validation of the model performed using multicenter data (n = 51). Receiver operating characteristics (ROC) curves and calibration plots were used to evaluate the prediction accuracy of the nomogram. Decision curve analysis (DCA) was implemented to illustrate the practicability of the nomogram clinical application. Based on the nomogram, we established a web calculator to visualize the risk of lymphatic metastases. We further plotted Kaplan-Meier overall survival (OS) curves to compare the survival time of patients with and without lymphatic metastasis.ResultsIn this study, the nomogram was established based on six significant factors (survival time, race, T stage, M stage, surgery, and lung metastasis), which were identified for lymphatic metastasis in ES patients. The model showed significant diagnostic accuracy with the value of the area under the curve (AUC) was 0.743 (95%CI: 0.714–0.771) for SEER internal validation and 0.763 (95%CI: 0.623–0.871) for multicenter data external validation. The calibration plot and DCA indicated that the model had vital clinical application value.ConclusionIn this study, we constructed and developed a nomogram with risk factors to predict lymphatic metastasis in ES patients and validated accuracy of itself. We found T stage (Tx OR = 2.540, 95%CI = 1.433–4.503, P < 0.01), M stage (M1, OR = 2.061, 95%CI = 1.189–3.573, P < 0.05) and survival time (OR = 0.982, 95%CI = 0.972–0.992, P < 0.001) were important independent factors for lymphatic metastasis in ES patients. Furthermore, survival time in patients with lymphatic metastasis or unclear situation (P < 0.0001) was significantly lower. It can help clinicians make better decisions to provide more accurate prognosis and treatment for ES patients.

Establishment and characterization of a novel patient-derived Ewing sarcoma cell line, NCC-ES2-C1.

Ewing sarcoma (ES) is a small round cell sarcoma that is characterized by the unique gene translocation EWSR1-FLI1. It is the second most common primary bone and soft tissue malignancy in children and adolescents. It constitutes 10-15% of all bone sarcomas and is highly aggressive and rapidly recurring. Although intensive treatments have improved the clinical outcome of ES patients, 20-25% of them exhibit metastases during diagnosis. Thus, the prognoses of these patients remain poor. Celllines are pivotal resources to investigate the molecular background of disease progression and to develop novel therapeutic modalities. In this study, we establishedand characterized a novel ES cell line, NCC-ES2-C1. The presence of the EWSR1-FLI1 fusion gene in these cells was confirmed in the NCC-ES2-C1 cells. Furthermore, these cells exhibited constant proliferation, and invasion, but did not form tumors in mice. We screened the anti-tumor effects of 214 anti-cancer drugs in NCC-ES2-C1 cells and found that the drugs which effectively reduced the proliferation of NCC-ES2-C1 cells. We concluded that NCC-ES2-C1 cells are a useful resource to study functions of the EWSR1-FLI1 fusion gene, investigate phenotypic changes caused by genes and proteins, and evaluate the anti-tumor effects of novel drugs.

Novel Targeted Therapeutic Strategies for Ewing Sarcoma.

Simple SummaryEwing sarcoma is an uncommon cancer that arises in mesenchymal tissues and represents the second most widespread malignant bone neoplasm after osteosarcoma in children. Therapy has increased the 5-year survival rate in the last 40 years, although the recurrence rate has remained high. There is an immediate and unmet need for the development of novel Ewing sarcoma therapies. We offer new prospective targets for the therapy of Ewing sarcoma. The EWSR1/FLI1 fusion protein, which is identified in 85–90% of Ewing sarcoma tumors, and its direct targets are given special focus in this study. Experimantal therapy that targets multiple signaling pathways activated during ES progression, alone or in combination with existing regimens, may become the new standard of care for Ewing sarcoma patients, improving patient survival.Ewing sarcoma (ES) is an uncommon cancer that arises in mesenchymal tissues and represents the second most widespread malignant bone neoplasm after osteosarcoma in children. Amplifications in genomic, proteomic, and metabolism are characteristics of sarcoma, and targeting altered cancer cell molecular processes has been proposed as the latest promising strategy to fight cancer. Recent technological advancements have elucidated some of the underlying oncogenic characteristics of Ewing sarcoma. Offering new insights into the physiological basis for this phenomenon, our current review examines the dynamics of ES signaling as it related to both ES and the microenvironment by integrating genomic and proteomic analyses. An extensive survey of the literature was performed to compile the findings. We have also highlighted recent and ongoing studies integrating metabolomics and genomics aimed at better understanding the complex interactions as to how ES adapts to changing biochemical changes within the tumor microenvironment.

A Machine Learning-Based Predictive Model for Predicting Lymph Node Metastasis in Patients With Ewing's Sarcoma.

ObjectiveIn order to provide reference for clinicians and bring convenience to clinical work, we seeked to develop and validate a risk prediction model for lymph node metastasis (LNM) of Ewing’s sarcoma (ES) based on machine learning (ML) algorithms.MethodsClinicopathological data of 923 ES patients from the Surveillance, Epidemiology, and End Results (SEER) database and 51 ES patients from multi-center external validation set were retrospectively collected. We applied ML algorithms to establish a risk prediction model. Model performance was checked using 10-fold cross-validation in the training set and receiver operating characteristic (ROC) curve analysis in external validation set. After determining the best model, a web-based calculator was made to promote the clinical application.ResultsLNM was confirmed or unable to evaluate in 13.86% (135 out of 974) ES patients. In multivariate logistic regression, race, T stage, M stage and lung metastases were independent predictors for LNM in ES. Six prediction models were established using random forest (RF), naive Bayes classifier (NBC), decision tree (DT), xgboost (XGB), gradient boosting machine (GBM), logistic regression (LR). In 10-fold cross-validation, the average area under curve (AUC) ranked from 0.705 to 0.764. In ROC curve analysis, AUC ranged from 0.612 to 0.727. The performance of the RF model ranked best. Accordingly, a web-based calculator was developed (https://share.streamlit.io/liuwencai2/es_lnm/main/es_lnm.py).ConclusionWith the help of clinicopathological data, clinicians can better identify LNM in ES patients. Risk prediction models established in this study performed well, especially the RF model.

Development of a Machine Learning-Based Predictive Model for Lung Metastasis in Patients With Ewing Sarcoma.

BackgroundThis study aimed to develop and validate machine learning (ML)-based prediction models for lung metastasis (LM) in patients with Ewing sarcoma (ES), and to deploy the best model as an open access web tool.MethodsWe retrospectively analyzed data from the Surveillance Epidemiology and End Results (SEER) Database from 2010 to 2016 and from four medical institutions to develop and validate predictive models for LM in patients with ES. Patient data from the SEER database was used as the training group (n = 929). Using demographic and clinicopathologic variables six ML-based models for predicting LM were developed, and internally validated using 10-fold cross validation. All ML-based models were subsequently externally validated using multiple data from four medical institutions (the validation group, n = 51). The predictive power of the models was evaluated by the area under receiver operating characteristic curve (AUC). The best-performing model was used to produce an online tool for use by clinicians to identify ES patients at risk from lung metastasis, to improve decision making and optimize individual treatment.ResultsThe study cohort consisted of 929 patients from the SEER database and 51 patients from multiple medical centers, a total of 980 ES patients. Of these, 175 (18.8%) had lung metastasis. Multivariate logistic regression analysis was performed with survival time, T-stage, N-stage, surgery, and bone metastasis providing the independent predictive factors of LM. The AUC value of six predictive models ranged from 0.585 to 0.705. The Random Forest (RF) model (AUC = 0.705) using 4 variables was identified as the best predictive model of LM in ES patients and was employed to construct an online tool to assist clinicians in optimizing patient treatment. (https://share.streamlit.io/liuwencai123/es_lm/main/es_lm.py).ConclusionsMachine learning were found to have utility for predicting LM in patients with Ewing sarcoma, and the RF model gave the best performance. The accessibility of the predictive model as a web-based tool offers clear opportunities for improving the personalized treatment of patients with ES.

GGAA-Microsatellites of NR0B1 Promoter Region in Ewing’s Sarcoma Patients and Healthy Individuals of a Southern Brazilian Population

Introduction: The very aggressive soft tissue and bone pediatric tumor Ewing’s sarcoma (ES) is caused in most cases by the chromosomal translocation t(11;22)(q24;q12), which encodes an aberrant chimeric transcription factor (EWS-FLI1) that regulates target genes, including the critical oncogene NR0B1 (Xp21.2), via GGAA microsatellites. Objective: Our objective was to analyze the GGAA microsatellites of NR0B1 promoter region of ES patients and healthy subjects in our population. Methodology: Ten male ES patients and 71 adult healthy males from Rio Grande do Sul state, Brazil, were included in this study. Leukocyte DNA was extracted, amplified by PCR, sequenced by the Sanger method and analyzed by capillary electrophoresis. Total number of GGAA motifs, length of microsatellite in base pairs, number of segments separated by "A" insertions and greatest number of consecutive GGAA motifs were analyzed. Statistical analyses were performed in the SPSS® statistical software and p-value &lt;0,05 was considered significant. Results: A total of 21 different alleles was identified in the 81 subjects, with 24.2 allele [(GGAA)7A(GGAA)7A(GGAA)10 sequence] being the most frequent, but when comparing the data between the two groups, no significant difference was found. Conclusion: We concluded that the studied sample was highly variable in terms microsatellite structure, including the presence of rare alleles, giving us the opportunity to describe our population which is a fundamental step on identifying genetic implications in ES tumorigenesis.

What Do We Know about Survival in Skeletally Premature Children Aged 0 to 10 Years with Ewing Sarcoma? A Multicenter 10-Year Follow-Up Study in 60 Patients.

Simple SummaryYounger age has been associated with better overall survival in Ewing sarcoma, especially under the age of 10. Our study aimed at describing long-term outcomes of a cohort of 60 patients aged 0–10 with Ewing sarcoma, treated with chemotherapy, surgery and/or radiotherapy. Overall survival of these youngest patients with ES was very good. After 10 years, 81% of patients were still alive, 89% did not have a local recurrence and 81% did not have distant metastasis (in lungs and/or bone). Limb salvage surgery was achieved in >90% of patients. Wide resection margin was the only factor significantly associated with better survival, but age < 6 years, smaller tumors, no metastases at diagnosis and treatment after 2000 also seemed to result in better overall survival.(1) Background: Younger age has been associated with better overall survival (OS) in Ewing sarcoma (ES), especially under the age of 10. The favorable survival in younger patients underlines the need for minimizing treatment burden and late sequelae. Our study aimed at describing clinical characteristics, treatment and outcome of a cohort of ES patients aged 0–10. (2) Methods: In this retrospective multicenter study, all consecutive ES patients aged 0–10, treated in four sarcoma centers in the Netherlands (n = 33) and one in Spain (n = 27) between 1982 and 2008, with a minimum follow-up of 10 years, were included. OS, local recurrence-free survival (LRFS) and distant metastasis-free survival (DMFS) were calculated. Potential factors of influence on OS (risk and protective factors) were analyzed. (3) Results: 60 patients with median follow-up 13.03 years were included. All patients were treated with chemotherapy in combination with local treatment, being surgery alone in 30 (50%) patients, radiotherapy (RT) alone in 12 (20%) patients or surgery plus RT in 18 (30%) patients (12 pre- and 6 postoperative). Limb salvage was achieved in 93% of patients. The 10-OS, -LRFS and -DMFS are 81% (95% CI: 71–91%), 89% (95% CI: 85–93%) and 81% (95% CI: 71–91%), respectively. Six patients developed LR, of which two developed subsequent DM; all had axial ES (pelvis, spine or chest wall), and these patients all died. Ten patients developed DM; eight died due to progressive disease, and two are currently in remission, both with pulmonary metastasis only. Negative or wide resection margin was significantly associated with better OS. Age < 6 years, tumor volume < 200 mL, absence of metastatic disease and treatment after 2000 showed trends towards better OS. Two patients developed secondary malignancy; both had chemotherapy combined with definitive RT for local treatment. (4) Conclusions: Overall survival of these youngest patients with ES was very good. Limb salvage surgery was achieved in >90% of patients. Wide resection margin was the only factor significantly associated with better survival.

Coupling Lipid Labeling and Click Chemistry Enables Isolation of Extracellular Vesicles for Noninvasive Detection of Oncogenic Gene Alterations.

Well‐preserved molecular cargo in circulating extracellular vesicles (EVs) offers an ideal material for detecting oncogenic gene alterations in cancer patients, providing a noninvasive diagnostic solution for detection of disease status and monitoring treatment response. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Here, a lipid labeling and click chemistry‐based EV capture platform (“Click Beads”), which is ideal for EV message ribonucleic acid (mRNA) assays due to its efficient, convenient, and rapid purification of EVs, enabling downstream molecular quantification using reverse transcription digital polymerase chain reaction (RT‐dPCR) is described and demonstrated. Ewing sarcoma protein (EWS) gene rearrangements and kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutation status are detected and quantified using EVs isolated by Click Beads and matched with those identified in biopsy specimens from Ewing sarcoma or pancreatic cancer patients. Moreover, the quantification of gene alterations can be used for monitoring treatment responses and disease progression.

Feasibility and safety of outpatient administration of ifosfamide and etoposide for pediatric patients with Ewing sarcoma in a resource-limited setting amid the COVID-19 pandemic.

BackgroundTo assess feasibility and safety of outpatient administration of ifosfamide and etoposide (IE) for pediatric Ewing sarcoma (EWS) patients in a resource‐limited setting amid the COVID‐19 pandemic.Materials and MethodsRetrospective study of patients with EWS who received outpatient IE from January 2020 until January 2021 at our institution. Ifosfamide 1800 mg/m2 was given for 5 days with MESNA (2‐mercaptoethane sulfonate sodium) infusion and additional boluses before and after 9 hours of hydration. Patients >10 years of age with the ability to drink orally at home, no pre‐existing renal dysfunction or history of hematuria were included. They were monitored for hemorrhagic cystitis with a urine dipstick before, midway, and at end of infusion. A urine analysis was done 24 hours after the last dose of ifosfamide.ResultsForty‐seven (100%) cycles were given as outpatient during the study period. Thirty‐five (74%) cycles were given on time, two (4%) cycles were delayed due to mucositis, two (4%) due to delayed count recovery, and eight (18%) due to unavailability of outpatient appointments. The median interval between these cycles was 15 days (range 14–44 days). No episode of hemorrhagic cystitis was reported. Urine analysis was not done at 24 hours for 27 (58%) cycles, so microscopic hematuria may have been missed. This outpatient protocol saved 32% (USD 299) per cycle in cost and 282 days of hospitalization.ConclusionOutpatient administration of IE for pediatric patients with EWS is feasible, safe, and cost‐effective during the COVID‐19 pandemic.