Ewing Sarcoma Patients Research Articles

Abstract 1175: Identification of novel combination therapies with the LSD1 inhibitor SP-2577 (Seclidemstat) in Ewing's Sarcoma using a complete druggable CRISPR library approach

Abstract Introduction: Ewing's Sarcoma (ES) is a rare and aggressive pediatric bone tumor that is driven by a chromosomal translocation and fusion between EWS and FLI1 genes resulting in a chimeric protein. Nearly 85% of ES cases express the EWS/FLI protein which functions as a transcription factor and drives oncogenesis. It has been previously observed in ES patient samples have high levels of the histone lysine specific demethylase 1 (LSD1). LSD1 is recruited by EWS/FLI as a member of the nucleosome remodeling and histone deacetylase (NuRD) complex to repress the critical EWS/FLI targets LOX and TGFBR2. Treatment with the potent LSD1 reversible inhibitor, SP-2577 (Seclidemstat), causes de-repression of these genes and impaired tissue culture cell viability in multiple ES cell lines. In this study we investigated new targeted therapies to use in combination with SP-2577 to help increase the cytotoxicity of the drug within ES cell types. Methods: Synthego® arrayed CRISPR screening platform was used to knock down genes associated with known druggable interactions in the ES cell line SK-N-MC. Genes were knocked down singularly either in the presence/absence of SP-2577. Viability screening was used to determine genes that increased the cytotoxicity of SP-2577. Statistical analysis, z-score, and IPA analysis was performed to investigate correlations between loss of gene effect and LSD1 pathway. Hit-gene validation was performed using siRNA and/or sgRNA knockdown/knockout, and other methods such as qPCR, and western blot assay for protein expression. Results: Arrayed CRISPR screening identified 103 gene deletions that resulted in increased SP-2577 cytotoxicity in SK-N-MC cell line. Among those, the top 19 hits were selected by identifying a FDA approved drug-gene interaction. Two high- interest hits were seen to have significant interaction with the LSD1 pathway: ABL2 and SH2B3. Drug combination treatment of SP-2577 with either Ruxolitinib or Dasatinib in ES cell lines showed high synergy at low dose ratio. Downstream pathways analysis confirmed the efficacy of the drugs combination. Detailed data will be presented. Conclusion: Our data support the validity of the arrayed CRISPR screening to identify new therapeutic strategies in ES, and highlights the potential for Ruxolitinib or Dasatinib treatment in combination with SP-2577 in ES patients. Citation Format: Samuel Sampson, Trason Thode, Mohan Kaadige, Alexis Weston, Serina Ng, Kevin Drenner, Raffaella Soldi, Sunil Sharma. Identification of novel combination therapies with the LSD1 inhibitor SP-2577 (Seclidemstat) in Ewing's Sarcoma using a complete druggable CRISPR library approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1175.

Role of High Dose Chemotherapy and Autologous Stem Cell Transplant in Relapsed Ewing's Sarcoma — a Systematic Review

Introduction: Relapsed Ewing's sarcoma (ES) is an aggressive malignancy with poor survival. Conventional dose salvage chemotherapy (CC) regimens given at the time of relapse led to response rate up to 28-68% with reported event free survival (EFS) of 29% at 2 years and Overall survival (OS) of 49 % at 1 year and 28-31 % at 2 years (Van Winkle, P. et al. 2005, Magnan et al. 2015). High dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) given as induction or as part of consolidation treatment after few cycles of CC has shown benefit in improving OS and EFS in single center studies. (McTiernan et al. 2006, Barker et al. 2005) Despite report of its utility, HDCT is not generally used in United states for relapsed ES and a comprehensive search and systemic review was performed to evaluate the benefits of HDCT for this disease.Methods: Comprehensive literature search was conducted in Medline (PubMed and Ovid SP), Embase and Cochrane Database of Systematic Review (CDSR). These databases were searched from the inception of databases till present. In addition, we manually selected more studies from bibliography data. We included studies that had relapsed Ewing's sarcoma patients and those which had mixed data for relapsed as well as primary metastatic disease treated with HDCT.Results: Through literature and bibliography search, 1005 studies were identified and after screening, 24 studies containing a total of 1459 patients were included in the final analysis. Seventeen studies had patients with primary metastatic disease and relapsed Ewing's sarcoma. Seven had only relapsed ES patients. In the studies with relapsed patients (n = 809), EFS for most studies at two and five years ranged from 42-44% and 24-43% respectively. The OS at two years and three to five years ranged from 50-66% and 21-50% respectively. In studies with combined primary metastatic and relapsed patients (n=650), EFS at one to two years and three to five years was 45-60% and 17-47% respectively. The OS at one to two years and three to five years was 40-70% and 31-50% respectively.Five studies (n=709) (Table 1) compared results of patients who received HDCT along with ASCT after CC to those who received only CC. Two to three and four to five years EFS for patients who received HDCT was 39-44% and 17-61% respectively, whereas it was 10-32% and 2-7% for patients who received only conventional dose therapy. OS of patients who received HDCT was 50-66% at two to three years and 42-77% at four to five years, whereas it was 14-34% and 7-10% respectively for patients who received only CC. Patients selected to receive HDCT in most of these studies were those who showed a response to initial CC (chemosensitive), either complete or partial response (CR/PR) (Rasper et al. 2014, Dirkson et al. 2014, Barker et al. 2005, Al faris et al. 2007, Frolich et al. 1999).Relapsed patients in Pole et al. (1984) survived only for three to six months. They had metastatic disease at relapse and did not receive conventional therapy prior to HDCT, possibly contributing to their decreased survival. Shankar et al 2003 reported EFS and OS at three and half years to be 14% and 28% respectively in spite of receiving CC followed by HDCT and ASCT.Conclusions: Most studies showed that HDCT followed by ASCT given as consolidation regimen improved OS and EFS compared to CC. Two studies (Pole et al. 1984 and Shankar et al. 2003) showed lower survival after HDCT when compared to CC. Patients in Pole et al study had metastatic disease at relapse and also did not receive CC prior to HDCT, which may have affected the survival. The reason for decreased survival in Shankar et al study is not clear. Five studies which compared patients who received HDCT and ASCT in addition to CC with patients who received only CC showed improved survival in patients who received HDCT followed by ASCT. Patients selected to receive HDCT showed initial chemosensitivity (by achieving CR or PR) to CC, suggesting that proof of chemosensitive disease is an important prognostic factor for improving survival with HDCT. The heterogeneity in the data prevented us from conducting a meta-analysis. Large scale randomized control studies are needed to determine true clinical benefit of HDCT followed by ASCT in patients with relapsed Ewing's sarcoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Whole Lung Irradiation after High-Dose Busulfan/Melphalan in Ewing Sarcoma with Lung Metastases: An Italian Sarcoma Group and Associazione Italiana Ematologia Oncologia Pediatrica Joint Study.

Simple SummaryThe lung is the most frequent site of metastasis in Ewing sarcoma, the second most common bone cancer affecting children, adolescents and young adults. The five-year overall survival of patients with isolated lung metastasis is approximately 50% after multimodal treatments including chemotherapy, surgery and radiotherapy. This retrospective study aimed to investigate the feasibility and the predictors of survival in 68 Ewing sarcoma patients with lung metastases who received high-dose chemotherapy with busulfan and melphalan, followed by reduced dose whole-lung irradiation, as part of two prospective and consecutive treatment protocols. This combined treatment strategy is feasible and might contribute to the disease control in lung metastatic Ewing sarcoma with responsive disease. Furthermore, the results of this study provide support to explore the treatment stratification for lung metastatic Ewing sarcoma based on the histological response of the primary tumor.Purpose: To analyze toxicity and outcome predictors in Ewing sarcoma patients with lung metastases treated with busulfan and melphalan (BU-MEL) followed by whole-lung irradiation (WLI). Methods: This retrospective study included 68 lung metastatic Ewing Sarcoma patients who underwent WLI after BU-MEL with autologous stem cell transplantation, as part of two prospective and consecutive treatment protocols. WLI 12 Gy for <14 years old and 15 Gy for ≥14 years old patients were applied at least eight weeks after BU-MEL. Toxicity, overall survival (OS), event-free survival (EFS) and pulmonary relapse-free survival (PRFS) were estimated and analyzed. Results: After WLI, grade 1–2 and grade 3 clinical toxicity was reported in 16.2% and 5.9% patients, respectively. The five-year OS, EFS and PRFS with 95% confidence interval (CI) were 69.8% (57.1–79.3), 61.2% (48.4–71.7) and 70.5% (56.3–80.8), respectively. Patients with good histological necrosis of the primary tumor after neoadjuvant chemotherapy showed a significant decreased risk of pulmonary relapse or death compared to patients with poor histological necrosis. Conclusions: WLI at recommended doses and time interval after BU-MEL is feasible and might contribute to the disease control in Ewing sarcoma with lung metastases and responsive disease. Further studies are needed to explore the treatment stratification based on the histological response of the primary tumor.

Prognostic Immune-Related Genes of Patients With Ewing's Sarcoma.

Ewing’s sarcoma (ES) is an extremely aggressive malignant bone tumor with a high incidence among children and adolescents. The immune microenvironment plays an important role in ES development. The aim of the current study was to investigate the immune microenvironment in ES patients to identify immune-related gene signatures. Single-sample gene set enrichment analysis (ssGSEA) was used to cluster the RNA sequences of 117 ES patients, and their immune cell infiltration data were downloaded and evaluated based on the Gene Expression Omnibus (GEO) database. High, medium, and low immune cell infiltration clusters were identified. Based on the comparison of clusters with high and low immune cell infiltration, normal skeletal muscle cells, and ES, we identified 198 common differentially expressed genes. GO and KEGG enrichment analyses indicated the underlying immune mechanism in ES. Cox and LASSO regression analyses were conducted to select immune-related prognostic genes. An external dataset from the International Cancer Genome Consortium (ICGC) was used to validate our results. Ten immune-related, independent prognostic genes (FMO2, GLCE, GPR64, IGFBP4, LOXHD1, PBK, SNAI2, SPP1, TAPT1-AS1, and ZIC2) were selected for analysis. These 10 immune-related genes signature were determined to exhibit independent prognostic significance for ES. The results of this study provide an approach for predicting the prognosis and survival of ES patients, and the elucidated genes may be a promising target for immunotherapy.

Comparison of treatment effect and long-term outcomes for pediatric and adult Ewing sarcoma patients in British Columbia, Canada.

11531 Background: Treatment of Ewing Sarcoma (EWS) is challenging. While it is known to be sensitive to chemotherapy and radiation, the number of lines of therapy available are limited. This disease affects pediatric patients (PP) more often than adults (AP), and reported outcomes are worse for AP onset EWS in the literature. It is unclear if this is due to difference in the biology of disease in AP compared to PP, or if this is due to differences in treatment approach. Furthermore, optimal treatments and real world impact of treatment is unclear in both the PP and AP populations. This study identifies the features of therapy received by AP and PP with EWS in a large, mutli-institutional cohort, and provides real world evidence for the expected outcomes in both AP and PP with EWS. Methods: A cohort study analysis of the Sarcoma Outcomes Unit database at BC Cancer was conducted to identify patients diagnosed with EWS in British Columbia from January 1, 2000 to December 31, 2018. Data on the frequency, amount, and regimen of chemotherapy were collected. Baseline Charlson comorbidity index, age at diagnosis, progression free survival and overall survival were collected. Results: 108 patients with EWS were identified, 66 AP and 42 PP Median age at diagnosis for adults was 37 (19-86) and median age for diagnosis of pediatric patients was 14 (1-18). Real world median PFS and OS for AP were 23 mos and 79 mos, and for the PP were 32 mos and NE. Five year overall survival was 54% in AP and 77% in PP. Overall, there was no difference in the number of lines of therapy received between PP and AP, but the type of therapy was more dose-dense in the PP than in the AP, (85% vs 28% for dose dense chemo). 5 year overall survival was longer for PP who received dose dense regimens compared to non-dose dense regimens (HR 0.87), but was not different in the AP receiving dose dense regimens (HR 0.95), even when controlling for comorbidities. The most common chemotherapy regimen for AP was Vincristine, Adriamycin, cyclophosphamide alternating with Ifosfamide and Etoposide q3weeks, whereas in the pediatric population the most common chemotherapy regimen was the same but alternating q2weeks. Conclusions: The treatment plans for PP with EWS were more often dose dense compared to the AP. Outcomes for PP were vastly better than for APs, despite overall similarities in the number of lines of therapy and types of agents used. Given the lack of difference between dose dense and non-dose dense regimens for APs, this is not the likely cause of difference in survival between PPs and APs. Extrapolating pediatric protocols to the adult setting may not be appropriate given the differences in outcomes. Further work to identify effective therapies and predictive biomarkers in this disease are needed, and my further identify reasons for discrepant outcomes in pediatric and adult populations.

Efficacy of dose intensification in induction therapy for localized Ewing sarcoma: Italian Sarcoma Group (ISG) and Associazione Italiana Ematologia ed Oncologia Pediatrica (AIEOP) ISG/AIEOP EW-1 study.

11501 Background: The role of dose intensification of chemotherapy in Ewing sarcoma (ES) is under evaluation in prospective trials. This is a controlled, randomized phase III study evaluating the impact on event-free survival (EFS) of two arms at different intensity of induction therapy in localized ES at onset. Methods: Newly diagnosed localized ES patients aged 2-40 were eligible. They were randomized to receive 4-courses induction therapy - 1 every 21 days - either with a standard arm (arm A) as per ISG/SSGIII protocol (Ferrari S, et at, Ann Oncol. 2011;22(5):1221) or with an intense arm B, consisting of vincristine 1,5mg/sqm+ doxorubicin 80mg/sqm+ifosfamide 9g/sqm for each course. After induction, patients underwent surgery and/or radiotherapy,followed by an adaptive treatment. Good responders received standard courses chemotherapy: arm A pts received 9 courses, while arm B pts received 5 courses. Poor responders in both arms received 4 courses followed by high-dose busulfan/melphalan+autologous stem cell rescue. The primary outcome measure was EFS for the 2 arms in the intention-to-treat population. Kaplan-Meier curves compared with log-rank test and Cox model were performed to assess differences between study arms. A secondary outcome was toxicity differences, assessed by means of the Fisher’s exact test. Initial sample size was 230 pts, type I error rate 5%, power 80%. Results: Between 2009 and 2019, 234 patients were randomized (arm A-115; arm B-119). M:F ratio was 1.8; median age 14 years (range 2-40); tumour site extremity in 55%, axial/pelvis in 45%; tumour volume &lt; 200ml in 31% and ≥200ml in 69%. A good response was obtained in 56% in arm A and 60% in arm B. Median follow-up was 68 months. EFS was not significantly different between arms; HR: 0.85; 95% CI: 0,51-1,41, 5-year EFS (95% CI) was 73% (64-82%) in arm A and 75% (67-83%) in arm B ( p = 0.526). Good responders in arm A and in arm B and poor responders in arm B had comparable results: 5-year EFS (95% CI) was 80% (71-91%), 77% (67-88%), and 72% (59-86%), respectively, while poor responders in arm A showed a worse, not statistically significant (p = 0.164) performance (63%; 50-78%). Subgroup analyses showed similar outcome for age, tumour site and volume in both arms. Hematological, gastrointestinal, and cardiovascular grade ≥3 toxicities were more pronounced in arm B (p &lt; 0.05). Conclusions: Intense induction therapy with arm B did not improve 5-year EFS when compared with the standard arm A. The higher toxicity observed in arm B than in arm A was counterbalanced, in good responders, by a similar outcome with a shorter treatment plan. For poor responders, with almost 30 patients per arm event-free and with &lt; 48-month FUP, better 5-year EFS in arm B than in arm A was observed but needs further observation. Clinical trial information: NCT02063022.

The MGMT (O6-methylguanine–DNA methyltransferase) promotes methylation and clinical outcomes of salvage temozolomide and irinotecan chemotherapy in progressive Ewing sarcoma: A preliminary report.

e23507 Background: There remains an unmet need to identify prognostic and predictive molecular biomarkers in advance Ewing sarcoma (ES). We sought to assess the influence of MGMT promoter methylation status on response rate, time to progression (TTP), and overall survival (OS) following salvage irinotecan and temozolomide (IT) chemotherapy. Methods: Data of advanced ES patients, treated with IT chemotherapy from Jan, 2014 to Jan, 2020 were retrospectively collected. Patients were required to have previously received and progressed after vincristine, doxorubicin, and cyclophosphamide alternating with ifosfamide and etoposide (VDC-IE). MGMT promoter methylation status was assessed by Methylation Sensitive Restriction Enzyme quantitative-PCR (MSRE-qPCR) on non-decalcified Formalin-fixed paraffin embedded (FFPE) tissue using internally developed primers and the OneStep qMethyl Kit (ZYMO RESEARCH CORP). Responses were assessed by response evaluation criteria in solid tumors (RECIST v. 1.1). TTP and OS were assessed by the Kaplan-Meier method. Survival comparisons were performed by the Log-rank test. Results: Herein, we present data of the preliminary analysis of the first 18 patients who underwent MGMT promoter methylation testing. Patients had a median age of 18 years (range: 5-34 years), and were predominantly male ( n=11; 61%). The primary tumor was located in the pelvis in 9 patients (50%), femur in 3 (17%), tibia in 2 (11%), kidney in 2 (11%), chest wall in one (6%), and scapula in one patient (6%). IT was given in a second ( n=15) or third-line setting ( n=3). At the time of initiation of IT, 13 patients (72%) had distant metastasis, and 5 (28%) had unresectable local progression in the pelvis ( n=4) or chest wall ( n=1). The mean percentage of MGMT promoter methylation was 29% (range: 3-98%). Five patients (28%) had methylated MGMT promoter, whereas the remaining had partially methylated ( n=6; 33%) or unmethylated ( n=7;39%) promoter. Five patients (28%) had objective response, with no observed difference according to MGMT promoter methylation ( p=0.58 for comparison between methylated and unmethylated/ partially methylated). Median TTP was 4.9 and 2.2 months for patients with methylated and partially methylated/ unmethylated MGMT respectively; p=0.76. The corresponding median OS was 69.4 and 14.3 months in favor of the methylated group; p=0.3. Conclusions: This preliminary data suggests a possible prognostic role for MGMT promoter methylation, with a markedly extended median OS for the methylated group. Nevertheless, the median OS difference did not reach statistical significance in this preliminary analysis. We plan to report data of the final analysis after finalizing MGMT testing for the rest of our study patients.

Prognostic significance of survivin expression in pediatric ewing sarcoma

Survivin is an inhibitor of apoptosis protein that inhibits caspases and blocks cell death. It is undetectable in most normal adult tissues. High survivin expression has been detected in various tumors and has been correlated with therapy resistance and poor outcome. We conducted this study to examine survivin expression in pediatric Ewing sarcoma (ES) and evaluate its role in predicting clinical outcome. Formalin-fixed paraffin-embedded tumor tissues from 108 pediatric ES patients were examined by immunostaining with survivin rabbit monoclonal antibodies. Survivin was detected in tumor tissues of 72 (66.7%) patients. High expression (≥50%) was detected in 18 (16.7%) patients. High survivin expression was shown to be a significant univariate parameter for poorer overall (OS) and event free survival (EFS) with p value 0.033, and 0.037 respectively. It was confirmed as an independent factor in multivariate analysis for OS (p: 0.041; HR: 1.97 with 95% CI of 1.03-3.79) and EFS (p: 0.049; HR: 1.86 with 95% CI of 1.00-3.46). These results suggest that high survivin expression identifies a group of patients with poor prognosis and this may help to refine risk adapted treatment; however, this needs to be confirmed in prospective studies.

Adolescent-adult nonmetastatic Ewing sarcoma-Experience from a large developing country.

Outcome and toxicity data in adolescent-adult Ewing sarcoma (AA-ES) patients are sparse and merits exploration. Histopathologically confirmed, nonmetastatic AA-ES patients, who received standard institutional combination chemotherapy regimen (Ewing's family of tumors-2001 [EFT-2001]) comprising of ifosfamide plus etoposide and vincristine, doxorubicin plus cyclophosphamide, lasting a total of 12months between 2013 and 2018, were analyzed for treatment-related toxicities, event-free survival (EFS), and overall survival (OS). There were 235 patients (primary safety cohort [PSC]) with median age of 23 (15-61) years; 159 (67.7%) were males, 155 (65.9%) had skeletal primary and 114 (48.5%) had extremity tumors. One hundred ninety-six (83.4%) were treatment naïve (primary efficacy cohort [PEC]) and of these 119 (60.7%) had surgery. In PEC, at a median follow-up of 36.4 (interquartile range [IQR] 20-55) months, estimated 3-year EFS and OS were 67.3% (95% CI 60.3-75.1%) and 91.1% (95% CI 86.7-95.7%), respectively. Of these, 158 (80.6%) complying with intended treatment, at a median follow-up of 39 (IQR 26-57) months had an estimated 3-year EFS of 68.2% (95% CI 60.3-76.1%). In multivariable analysis, good prognostic factors included longer symptom(s) duration (HR 0.93, 95% CI 0.86-0.994), ≥99% necrosis (HR 0.30, 95% CI 0.11-0.77), and treatment completion (HR 0.32, 95% CI 0.14-0.74). Among PSC, grade 3-4 toxicities were febrile neutropenia (119, 50.6%), anemia (130, 55.3%), peripheral neuropathy (37, 15.7%), with three (1.3%) chemo-toxic deaths. The outcomes of AA nonmetastatic ES patients treated with EFT-2001 regimen were comparable to those reported by others, with acceptable toxicity. This regimen can be considered a standard of care in AA-ES.

Circulating Plasma Tumor DNA Is Superior to Plasma Tumor RNA Detection in Ewing Sarcoma Patients: ptDNA and ptRNA in Ewing Sarcoma

The detection of tumor-specific nucleic acids from blood increasingly is being used as a method of liquid biopsy and minimal residual disease detection. However, achieving high sensitivity and high specificity remains a challenge. Here, we perform a direct comparison of two droplet digital PCR (ddPCR)-based detection methods, circulating plasma tumor RNA and circulating plasma tumor DNA (ptDNA), in blood samples from newly diagnosed Ewing sarcoma patients. First, we developed three specific ddPCR-based assays to detect EWS-FLI1 or EWS-ERG fusion transcripts, which naturally showed superior sensitivity to DNA detection on invitro control samples. Next, we identified the patient-specific EWS-FLI1 or EWS-ERG breakpoint from five patient tumor samples and designed ddPCR-based, patient-specific ptDNA assays for each patient. These patient-specific assays show that although plasma tumor RNA can be detected in select newly diagnosed patients, positive results are low and statistically unreliable compared with ptDNA assays, which reproducibly detect robust positive results across most patients. Furthermore, the unique disease biology of Ewing sarcoma enabled us to show that most cell-free RNA is not tumor-derived, although cell-free-DNA burden is affected strongly by tumor-derived DNA burden. Here, we conclude that, even with optimized highly sensitive and specific assays, tumor DNA detection is superior to RNA detection in Ewing sarcoma patients.

Current Status of Management and Outcome for Patients with Ewing Sarcoma.

Simple SummaryEwing sarcoma is a highly aggressive malignancy affecting primarily children and adolescents. It is the second most common bone sarcoma among children, affecting between 1 and 3 persons per million inhabitants. The tumor typically carries a pathognomonic chromosomal translocation resulting in a fusion transcript (EWSR1-FLI1), which plays an orchestral role in tumor development. While the fusion transcript has been known for decades, targeted treatment has been disappointing. However, new molecular techniques such as next-generation sequencing have significantly increased our understanding of this rare disease. Moreover, new closely related tumors with similar morphology but different characteristics have evolved. Treatment today consists of multiagent chemotherapy, radiation treatment, and surgery, all of which are associated with significant short- and long-term side effects. In this review article, we describe the currently existing diagnostic- and treatment-related challenges as well as the most important ongoing or recently conducted studies.Ewing sarcoma is the second most common bone sarcoma in children after osteosarcoma. It is a very aggressive malignancy for which systemic treatment has greatly improved outcome for patients with localized disease, who now see survival rates of over 70%. However, for the quarter of patients presenting with metastatic disease, survival is still dismal with less than 30% of patients surviving past 5 years. Patients with disease relapse, local or distant, face an even poorer prognosis with an event-free 5-year survival rate of only 10%. Unfortunately, Ewing sarcoma patients have not yet seen the benefit of recent years’ technical achievements such as next-generation sequencing, which have enabled researchers to study biological systems at a level never seen before. In spite of large multinational studies, treatment of Ewing sarcoma relies entirely on chemotherapeutic agents that have been largely unchanged for decades. As many promising modern therapies, including monoclonal antibodies, small molecules, and immunotherapy, have been disappointing to date, there is no clear candidate as to which drug should be investigated in the next large-scale clinical trial. However, the mechanisms driving tumor development in Ewing sarcoma are slowly unfolding. New entities of Ewing-like tumors, with fusion transcripts that are related to the oncogenic EWSR1-FLI1 fusion seen in the majority of Ewing tumors, are being mapped. These tumors, although sharing much of the same morphologic features as classic Ewing sarcoma, behave differently and may require a different treatment. There are also controversies regarding local treatment of Ewing sarcoma. The radiosensitive nature of the disease and the tendency for Ewing sarcoma to arise in the axial skeleton make local treatment very challenging. Surgical treatment and radiotherapy have their pros and cons, which may give rise to different treatment strategies in different centers around the world. This review article discusses some of these controversies and reproduces the highlights from recent publications with regard to diagnostics, systemic treatment, and surgical treatment of Ewing sarcoma.

The Controversial Role of Autophagy in Ewing Sarcoma Pathogenesis-Current Treatment Options.

Ewing Sarcoma (ES) is a rare, aggressive, and highly metastasizing cancer in children and young adults. Most ES cases carry the fusion of the Ewing Sarcoma Breakpoint Region 1 (EWSR1) and FLI1 (Friend leukemia virus integration site 1) genes, leading to an EWS–FLI1 fused protein, which is associated with autophagy, a homeostatic and catabolic mechanism under normal and pathological conditions. Following such interesting and controversial data regarding autophagy in ES, many clinical trials using modulators of autophagy are now underway in this field. In the present review, we summarize current data and clinical trials that associate autophagy with ES. In vitro studies highlight the controversial role of autophagy as a tumor promoter or a tumor suppressor mechanism in ES. Clinical and in vitro studies on ES, together with the autophagy modulators, suggest that caution should be adopted in the application of autophagy as a therapeutic target. Monitoring and targeting autophagy in every ES patient could eliminate the need for targeting multiple pathways in order to achieve the maximum beneficial effect. Future studies are required to focus on which ES patients are affected by autophagy modulators in order to provide novel and more efficient therapeutic protocols for patients with ES based on the current autophagy status of the tumors.

The roles of metastasis-related proteins in the development of giant cell tumor of bone, osteosarcoma and Ewing's sarcoma.

BACKGROUND: Giant cell tumor of bone (GC), osteosarcoma (OS) and Ewing’s sarcoma (ES) are three different types of bone cancer with common and specific pathology features.OBJECTIVE: The purpose of the study was to examine the relationship and differences of the three bone tumors using clinical samples.METHODS: Through screening the profiles of clinical samples from GC, OS and ES patients using a humanoncology array, we found 26, 25 and 15 tumorigenesis factors significantly increased in GS, OS and ES tissues compared to normal individuals. eNOS, endostatin, HIF-1, IL-6, CCL2/MCP-1, CCL8/MCP-2, CCL7/MCP-3, Tie and VEGF directly or indirectly involve in the metastasis Therefore, expression levels of the 6 factors were further determined by Western blot.RESULTS: The results showed levels of MCP1, MCP2, MCP3 or IL-6 in the GS, OS and ES significantly increased, and the expression levels of angiogenesis and anti-angiogenesis factors containing eNOS, endostatin, HIF-1, Tie or VEGF were enhanced.CONCLUSIONS: Our results suggest that eNOS, endostatin, HIF-1, IL-6, CCL2/MCP-1, CCL8/MCP-2, CCL7/MCP-3, Tie and VEGF may play important roles in tumorigenesis, reveal the expression differences of tumor-associated cytokines and angiogenesis related factors, and provide clinical evidence for studying the mechanisms on the metastasis in GC, OS and ES.

Renal sarcomas: Epidemiology, treatment and outcomes.

362 Background: Renal sarcomas are a rare malignancy in adults and have been inadequately evaluated on a US national level regarding epidemiology, treatment, and outcomes. Methods: The 2004-2016 NCDB and SEER databases were queried for adult patients diagnosed with sarcomas of renal origin. Age-adjusted incidence rates were derived from the SEER database. Overall survival (OS) was assessed using multivariable Cox proportional hazards models adjusting for demographics, tumor and treatment variables. Results: 1,279 renal sarcomas comprising 39 subtypes were reported from 2004-2016, contributing 0.3% of all NCDB renal malignancies. As shown in the table below, the most common subtypes were leiomyosarcoma (LMS), angiosarcoma (AS), malignant rhabdoid tumor (MRT), dedifferentiated liposarcoma (DL) and primitive neuroectodermal tumors (PNET). Over the study period, renal sarcoma incidence rates remained constant at 0.5 cases / 1 million citizens. Sex-specific incidence differences were evident with female predominance for LMS, and male predominance for AS. Age at diagnosis and tumor diameter varied according to sarcoma subtypes: for example, median age in LMS was 62y compared to 30y in Ewing sarcoma patients; median tumor diameter was 18cm for solitary fibrous tumors and 7.5cm for synovial sarcoma. Renal sarcoma was staged as T3 in 33.3% and T4 in 14.2%, while distant metastases were evident in 29.1% of cases at diagnosis. Most T1-T3 stage renal sarcomas underwent surgical resection (992/1098, 84%), compared to 71% for T4 renal sarcomas (128/181). Systemic therapy was administered in 32.1% of renal sarcoma cases (23.5% combined with surgical resection). Renal sarcoma 1-,2-, and 5-year OS rates were 48%, 24%, and 13%. OS was worse for T4 vs T1-3 sarcomas (HR=1.6, p&lt;0.001), and cases with distant metastases vs none (HR=3.2, p&lt;0.001). As summarized in the table, OS varied according to sarcoma subtypes with worse OS for AS compared to PNET (HR=1.5, p=0.04). Conclusions: Accounting for 0.3% of renal malignancies in adults, renal sarcomas include 39 different histological subtypes with distinct demographics, tumor parameters and outcomes. Renal sarcomas commonly present with advanced T stage at diagnosis and are treated with surgical resection with or without systemic therapy. [Table: see text]

No difference in survival after HLA mismatched versus HLA matched allogeneic stem cell transplantation in Ewing sarcoma patients with advanced disease

Patients with advanced Ewing sarcoma (AES) carry a poor prognosis. Retrospectively, we analyzed 66 AES patients treated with allogeneic stem cell transplantation (allo-SCT) receiving HLA-mismatched (group A, n = 39) versus HLA-matched grafts (group B, n = 27). Median age at diagnosis was 13 years, and 15 years (range 3–49 years) at allo-SCT. The two groups did not differ statistically in distribution of gender, age, remission status/number of relapses at allo-SCT, or risk stratum. 9/39 (23%) group A versus 2/27 (7%) group B patients developed severe acute graft versus host disease (GvHD). Of patients alive at day 100, 7/34 (21%) group A versus 9/19 (47%) group B patients had developed chronic GvHD. In group A, 33/39 (85%) versus 20/27 (74%) group B patients died of disease and 1/39 (3%) versus 1/27 (4%) patients died of complications, respectively. Altogether 12/66 (18%) patients survived in CR. Median EFS 24 months after allo-SCT was 20% in both groups, median OS was 27% (group A) versus 17% (group B), respectively. There was no difference in EFS and OS in AES patients transplanted with HLA-mismatched versus HLA-matched graft in univariate and multivariate analyses. In this analysis, CR at allo-SCT is a condition for survival (p < 0.02).

Systematic review of phase-I/II trials enrolling refractory and recurrent Ewing sarcoma: Actual knowledge and future directions to optimize the research.

BackgroundOptimal Phase‐II design to evaluate new therapies in refractory/relapsed Ewing sarcomas (ES) remains imperfectly defined.ObjectivesRecurrent/refractory ES phase‐I/II trials analysis to improve trials design.MethodsComprehensive review of therapeutic trials registered on five databases (who.int/trialsearch, clinicaltrials.gov, clinicaltrialsregister.eu, e‐cancer.fr, and umin.ac.jp) and/or published in PubMed/ASCO/ESMO websites, between 2005 and 2018, using the criterion: (Ewing sarcoma OR bone sarcoma OR sarcoma) AND (Phase‐I or Phase‐II).ResultsThe 146 trials identified (77 phase‐I/II, 67 phase‐II, and 2 phase‐II/III) tested targeted (34%), chemo‐ (23%), immune therapies (19%), or combined therapies (24%). Twenty‐three trials were ES specific and 48 had a specific ES stratum. Usually multicentric (88%), few trials were international (30%). Inclusion criteria cover the recurrent ES age range for only 12% of trials and allowed only accrual of measurable diseases (RECIST criteria). Single‐arm design was the most frequent (88%) testing mainly single drugs (61%), only 5% were randomized. Primary efficacy outcome was response rate (RR=CR+PR; Complete+Partial response) (n = 116/146; 79%), rarely progression‐free or overall survival (16% PFS and 3% OS). H0 and H1 hypotheses were variable (3%–25% and 20%–50%, respectively). The 62 published trials enrolled 827 ES patients. RR was poor (10%; 15 CR=1.7%, 68 PR=8.3%). Stable disease was the best response for 186 patients (25%). Median PFS/OS was of 1.9 (range 1.3–14.7) and 7.6 months (5–30), respectively. Eleven (18%) published trials were considered positive, with median RR/PFS/OS of 15% (7%–30%), 4.5 (1.3–10), and 16.6 months (6.9–30), respectively.ConclusionThis review supports the need to develop the international randomized phase‐II trials across all age ranges with PFS as primary endpoint.

P21-activated kinases as viable therapeutic targets for the treatment of high-risk Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults. Unfortunately, there have been minimal recent advancements in improving patient outcomes, especially in metastatic and recurrent diseases. In this study, we investigated the biological role of p21-activated kinases (PAKs) in ES, and the ability to therapeutically target them in high-risk disease. Via informatics analysis, we established the inverse association of PAK1 and PAK4 expression with clinical stage and outcome in ES patients. Through expression knockdown and small-molecule inhibition of PAKs, utilizing FRAX-597, KPT-9274, and PF-3758309 in multiple ES cell lines and patient-derived xenograft models, we further explored the role of PAKs in ES tumor growth and metastatic capabilities. In vitro studies in several ES cell lines indicated that diminishing PAK1 and PAK4 expression reduces tumor cell viability, migratory, and invasive properties. In vivo studies using PAK4 inhibitors, KPT-9274 and PF-3758309 demonstrated significant inhibition of primary and metastatic tumor formation, while transcriptomic analysis of PAK4-inhibitor-treated tumors identified concomitant suppression of Notch, β-catenin, and hypoxia-mediated signatures. In addition, the analysis showed enrichment of anti-tumor immune regulatory mechanisms, including interferon (IFN)-ɣ and IFN-α responses. Altogether, our molecular and pre-clinical studies are the first to establish a critical role for PAKs in ES development and progression, and consequently as viable therapeutic targets for the treatment of high-risk ES in the near future.

LncRNA TUG1 promotes Ewing's sarcoma cell proliferation, migration, and invasion via the miR-199a-3p-MSI2 signaling pathway.

The aim of this study was to investigate the roles and potential mechanisms of long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in the proliferation, migration, and invasion of Ewing's sarcoma cells. RT-qPCR was used to detect the expression of TUG1, microRNA-199a-3p (miR-199a-3p), and musashi2 (MSI2) in Ewing's sarcoma tissues and cell lines. Kaplan-Meier overall survival curves showed the survival rates of Ewing's sarcoma patients with high and low expression of TUG1. The association between the expressions of TUG1/MSI2 and miR-199a-3p in Ewing's sarcoma tissues was assessed by Pearson's correlation analysis. Cell proliferation, migration, and invasion were detected by CCK-8 assay and Transwell assay, respectively. The protein level of MSI2 was determined using western blotting. The interaction between TUG1/MSI2 and miR-199a-3p was validated by the dual-luciferase reporter assay. The levels of TUG1 and MSI2 were increased, while the level of miR-199a-3p was decreased in Ewing's sarcoma tissues and cells. High expression of TUG1 or MSI2 indicated a decreased overall survival rate of Ewing's sarcoma patients. TUG1/MSI2 level was negatively correlated with miR-199a-3p level. While TUG1 level was positively correlated with MSI2 level. In Ewing's sarcoma cells, knockdown of TUG1/MSI2 or overexpression of miR-199a-3p inhibited cell proliferation, migration, and invasion, whereas the overexpression of TUG1/MSI2 presented the opposite results. TUG1 functioned as a competing endogenous RNA to regulate MSI2 expression by sponging miR-199a-3p. Finally, miR-199a-3p inhibitor or MSI2 overexpression counteracted the TUG1 knockdown-mediated inhibitory effect on Ewing's sarcoma cell proliferation, migration, and invasion. TUG1 promotes proliferation, migration, and invasion of Ewing's sarcoma cells via sequestering miR-199a-3p to enhance the MSI2 expression, suggesting that TUG1 might be a potential target for treating Ewing's sarcoma.

Liquid Biopsies in Ewing Sarcoma.

Liquid biopsies enable noninvasive therapy monitoring in patients with solid tumors. Specific serum markers such as proteins, hormones, or enzymes released from tumor cells or in response to tumor growth can be used for quantification of the tumor burden. However, only a fraction of pediatric tumors has none of these serum markers, but tumor-specific genetic alterations represent reliable alternatives. Here we describe a method for using genomic fusion sequences as liquid biopsy markers in Ewing sarcoma patients.

Lung Metastasis Probability in Ewing Sarcoma: A Nomogram Based on the SEER Database.

Background. Up to now, an accurate nomogram to predict the lung metastasis probability in Ewing sarcoma (ES) at initial diagnosis is lacking. Our objective was to construct and validate a nomogram for the prediction of lung metastasis in ES patients. Methods. A total of 1157 patients with ES from the Surveillance, Epidemiology, and End Results (SEER) database were retrospectively collected. The predictors of lung metastasis were identified via the least absolute shrinkage and selection operator (LASSO) and multivariate logistic analysis. The discrimination and calibration of the nomogram were validated by receiver operating characteristic (ROC) curve and calibration curve. Decision curve analysis (DCA) was used to evaluate the clinical usefulness and net benefits of the prediction model. Results. Factors including age, tumor size, primary site, tumor extension, and other site metastasis were identified as the ultimate predictors for the nomogram. The calibration curves for the training and validation cohorts both revealed good agreement, and the Hosmer–Lemeshow test identified that the model was well fitted (p > 0.05). In addition, the area under the ROC curve (AUC) values in the training and validation cohorts were 0.732 (95% confidence interval, CI: 0.607–0.808) and 0.741 (95% CI: 0.602–0.856), respectively, indicating good predictive discrimination. The DCA showed that when the predictive metastasis probability was between 1% and 90%, the nomogram could provide clinical usefulness and net benefit. Conclusion. The nomogram constructed and validated by us could provide a convenient and effective tool for clinicians that can improve prediction of the probability of lung metastasis in patients with ES at initial diagnosis.