Pleuroparenchymal fibroelastosis as a late complication of childhood cancer therapy: A case series.

Pleuroparenchymal fibroelastosis (PPFE) is a rare interstitial pneumonia with distinct clinicopathologic features. It has been associated with exposure to hematopoietic stem cell transplantation (HSCT) and classical alkylating agents. Here, we highlight PPFE as a late complication of childhood cancer therapy by describing the cases of four survivors of childhood cancer with a diagnosis of treatment-related PPFE. All patients received high-dose alkylating agents. PPFE should be considered in the differential diagnosis of restrictive lung disease in patients with history of exposure to alkylating agents or HSCT. Development of PPFE-specific, noninvasive diagnostic tools and disease-modifying therapies will clinically benefit these patients.

Chromoplexy Is a Frequent Early Clonal Event in EWSR1-Rearranged Round Cell Sarcomas That Can Be Detected Using Clinically Validated Targeted Sequencing Panels.

Chromoplexy is a phenomenon defined by large-scale chromosomal chained rearrangements. A previous study observed chromoplectic events in a subset of Ewing sarcomas (ES), which was linked to an increased relapse rate. Chromoplexy analysis could potentially facilitate patient risk stratification, particularly if it could be detected with clinically applied targeted next-generation sequencing (NGS) panels. Using DELLY, a structural variant (SV) calling algorithm that is part of the MSK-IMPACT pipeline, we characterized the spectrum of SVs in EWSR1-fused round cell sarcomas, including 173 ES and 104 desmoplastic small round cell tumors (DSRCT), to detect chromoplexy and evaluate its association with clinical and genomic features. Chromoplectic events were detected in 31% of the ES cases and 19% of the DSRCT cases. EWSR1 involvement accounted for 76% to 93% of these events, being rearranged with diverse noncanonical gene partners across the genome, involving mainly translocations but also intrachromosomal deletions and inversions. A major breakpoint cluster was located on EWSR1 exons 8-13. In a subset of cases, the SVs disrupted adjacent loci, forming deletion bridges. Longitudinal sequencing and breakpoint allele fraction analysis showed that chromoplexy is an early event that remains detectable throughout disease progression and likely develops simultaneously with the driver fusion. The presence of chromoplexy was validated in an external ES patient cohort with whole exome sequencing. Chromoplexy was significantly more likely to be present in cases that were metastatic at presentation. Together, this study identifies chromoplexy as a frequent genomic alteration in diverse EWSR1-rearranged tumors that can be captured by targeted NGS panels. SIGNIFICANCE: Chromoplexy is detectable using targeted NGS in a substantial portion of EWSR1-rearranged round cell sarcomas as an early and persistent clonal event, expanding the genomic complexity of fusion-associated sarcomas.

Subclonal Somatic Copy-Number Alterations Emerge and Dominate in Recurrent Osteosarcoma.

Multiple large-scale genomic profiling efforts have been undertaken in osteosarcoma to define the genomic drivers of tumorigenesis, therapeutic response, and disease recurrence. The spatial and temporal intratumor heterogeneity could also play a role in promoting tumor growth and treatment resistance. We conducted longitudinal whole-genome sequencing of 37 tumor samples from 8 patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. Subclonal copy-number alterations were identified in all patients except one. In 5 patients, subclones from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clones in 6 of 7 patients with multiple clones. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy-number clones. A chromosomal duplication timing analysis revealed that complex genomic rearrangements typically occurred prior to diagnosis, supporting a macroevolutionary model of evolution, where a large number of genomic aberrations are acquired over a short period of time followed by clonal selection, as opposed to ongoing evolution. A mutational signature analysis of recurrent tumors revealed that homologous repair deficiency (HRD)-related SBS3 increases at each time point in patients with recurrent disease, suggesting that HRD continues to be an active mutagenic process after diagnosis. Overall, by examining the clonal relationships between temporally and spatially separated samples from patients with relapsed/refractory osteosarcoma, this study sheds light on the intratumor heterogeneity and potential drivers of treatment resistance in this disease. SIGNIFICANCE: The chemoresistant population in recurrent osteosarcoma is subclonal at diagnosis, emerges at the time of primary resection due to selective pressure from neoadjuvant chemotherapy, and is characterized by unique oncogenic amplifications.

Tandem LC-MS Identification of Antitubercular Compounds in Zones of Growth Inhibition Produced by South African Filamentous Actinobacteria.

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7-151.6 nM against M. tuberculosis H37RvTin vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.

Clinical Targeted Next-Generation Panel Sequencing Reveals MYC Amplification Is a Poor Prognostic Factor in Osteosarcoma.

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 TP53, 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.

Subclonal somatic copy number alterations emerge and dominate in recurrent osteosarcoma.

Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma.

Deep Learning-Based Objective and Reproducible Osteosarcoma Chemotherapy Response Assessment and Outcome Prediction.

Osteosarcoma is the most common primary bone cancer, whose standard treatment includes pre-operative chemotherapy followed by resection. Chemotherapy response is used for prognosis and management of patients. Necrosis is routinely assessed after chemotherapy from histology slides on resection specimens, where necrosis ratio is defined as the ratio of necrotic tumor/overall tumor. Patients with necrosis ratio ≥90% are known to have a better outcome. Manual microscopic review of necrosis ratio from multiple glass slides is semiquantitative and can have intraobserver and interobserver variability. In this study, an objective and reproducible deep learning-based approach was proposed to estimate necrosis ratio with outcome prediction from scanned hematoxylin and eosin whole slide images (WSIs). To conduct the study, 103 osteosarcoma cases with 3134 WSIs were collected. Deep Multi-Magnification Network was trained to segment multiple tissue subtypes, including viable tumor and necrotic tumor at a pixel level and to calculate case-level necrosis ratio from multiple WSIs. Necrosis ratio estimated by the segmentation model highly correlates with necrosis ratio from pathology reports manually assessed by experts. Furthermore, patients were successfully stratified to predict overall survival with P = 2.4 × 10-6 and progression-free survival with P = 0.016. This study indicates that deep learning can support pathologists as an objective tool to analyze osteosarcoma from histology for assessing treatment response and predicting patient outcome.

Irinotecan dose schedule for the treatment of Ewing sarcoma.

Irinotecan and temozolomide achieve objective responses in patients with Ewing sarcoma that recurs after initial therapy. Optimal dose schedules have not been defined. We reviewed published series of patients treated with irinotecan and temozolomide for Ewing sarcoma that recurred after initial therapy. We compared objective response rates for patients who received 5-day irinotecan treatment schedules to response rates for patients who achieved 10-day irinotecan treatment schedules. Among 89 patients treated with a 10-day irinotecan schedule, there were 47 objective responses (53%). Among 180 patients treated with a 5-day irinotecan schedule, there were 52 responses (29%). In the treatment of recurrent Ewing sarcoma, investigators should consider the use of a 10-day schedule for administration of irinotecan.

Oxalate utilisation is widespread in the actinobacterial genus Kribbella.

The type strains of all 33 species in the genus Kribbella were tested for growth on oxalate (-OOC-COO-) as sole carbon source. Media were initially formulated to contain sodium oxalate, but even a concentration as low as 7.5 mM oxalate prevented growth. A modified medium based on calcium oxalate was very successful in characterising oxalate utilisation by Kribbella strains (metabolism of oxalate by oxalotrophic bacteria results in visible zones of clearing around the growth streaks on the opaque plates). To assess the variability of oxalate utilisation in Kribbella species, we also tested eight non-type strains for their ability to use oxalate. Thirty of 33 type strains (90.9%) and six of eight non-type strains (75%) were able to use oxalate as a sole carbon source. Based on these results, we propose that oxalate would be an excellent carbon source for the selective isolation of Kribbella strains. Based on the oxalate-utilisation phenotype and analyses of the 19 publicly available Kribbella type-strain genome sequences, we propose a pathway for oxalate metabolism in Kribbella. This pathway is significantly different from those previously proposed for oxalate metabolism in other bacteria, involving the indirect catabolism of oxalate to formate. Formate production is proposed to be involved in energy generation and to be crucial for oxalate import via an oxalate:formate antiporter. To our knowledge, this is the first report of an oxalate:formate antiporter in an aerobic, Gram-positive bacterium.

Prospective Clinical Genomic Profiling of Ewing Sarcoma: ERF and FGFR1 Mutations as Recurrent Secondary Alterations of Potential Biologic and Therapeutic Relevance.

PURPOSE: Ewing sarcoma (ES) is a primitive sarcoma defined by EWSR1-ETS fusions as the primary driver alteration. To better define the landscape of cooperating secondary genetic alterations in ES, we analyzed clinical genomic profiling data of 113 patients with ES, a cohort including more adult patients (> 18 years) and more patients with advanced stage at presentation than previous genomic cohorts. METHODS: The data set consisted of patients with ES prospectively tested with the US Food and Drug Administration-cleared Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets large panel, hybrid capture-based next-generation sequencing assay. To assess the functional significance of ERF loss, we generated ES cell lines with increased expression of ERF and lines with knockdown of ERF. We assessed cell viability, clonogenic growth, and motility in these ES lines and performed transcriptomic and epigenetic analyses. Finally, we validated our findings in vivo using cell line xenografts. RESULTS: Novel subsets were defined by recurrent secondary alterations in ERF, which encodes an ETS domain transcriptional repressor, in 7% of patients (five truncating mutations, one deep deletion, and two missense mutations) and in FGFR1 in another 2.7% (one amplification and two known activating mutations). ERF alterations were nonoverlapping with STAG2 alterations. In vitro, increased expression of ERF decreased tumor cell growth, colony formation, and motility in two ES cell lines, whereas ERF loss induced cellular proliferation and clonogenic growth. Transcriptomic analysis of cell lines with ERF loss revealed an increased expression of genes and pathways associated with aggressive tumor biology, and epigenetic, chromatin-based studies revealed that ERF competes with EWSR1-FLI1 at ETS-binding sites. CONCLUSION: Our findings open avenues to new insights into ES pathobiology and to novel therapeutic approaches in a subset of patients with ES.

Clinical sequencing of soft tissue and bone sarcomas delineates diverse genomic landscapes and potential therapeutic targets.

The genetic, biologic, and clinical heterogeneity of sarcomas poses a challenge for the identification of therapeutic targets, clinical research, and advancing patient care. Because there are > 100 sarcoma subtypes, in-depth genetic studies have focused on one or a few subtypes. Herein, we report a comparative genetic analysis of 2,138 sarcomas representing 45 pathological entities. This cohort is prospectively analyzed using targeted sequencing to characterize subtype-specific somatic alterations in targetable pathways, rates of whole genome doubling, mutational signatures, and subtype-agnostic genomic clusters. The most common alterations are in cell cycle control and TP53, receptor tyrosine kinases/PI3K/RAS, and epigenetic regulators. Subtype-specific associations include TERT amplification in intimal sarcoma and SWI/SNF alterations in uterine adenosarcoma. Tumor mutational burden, while low compared to other cancers, varies between and within subtypes. This resource will improve sarcoma models, motivate studies of subtype-specific alterations, and inform investigations of genetic factors and their correlations with treatment response.

Extracorporeal Treatment for Methotrexate Poisoning: Systematic Review and Recommendations from the EXTRIP Workgroup.

Methotrexate is used in the treatment of many malignancies, rheumatological diseases, and inflammatory bowel disease. Toxicity from use is associated with severe morbidity and mortality. Rescue treatments include intravenous hydration, folinic acid, and, in some centers, glucarpidase. We conducted systematic reviews of the literature following published EXtracorporeal TReatments In Poisoning (EXTRIP) methods to determine the utility of extracorporeal treatments in the management of methotrexate toxicity. The quality of the evidence and the strength of recommendations (either "strong" or "weak/conditional") were graded according to the GRADE approach. A formal voting process using a modified Delphi method assessed the level of agreement between panelists on the final recommendations. A total of 92 articles met inclusion criteria. Toxicokinetic data were available on 90 patients (89 with impaired kidney function). Methotrexate was considered to be moderately dialyzable by intermittent hemodialysis. Data were available for clinical analysis on 109 patients (high-dose methotrexate [>0.5 g/m2]: 91 patients; low-dose [≤0.5 g/m2]: 18). Overall mortality in these publications was 19.5% and 26.7% in those with high-dose and low-dose methotrexate-related toxicity, respectively. Although one observational study reported lower mortality in patients treated with glucarpidase compared with those treated with hemodialysis, there were important limitations in the study. For patients with severe methotrexate toxicity receiving standard care, the EXTRIP workgroup: (1) suggested against extracorporeal treatments when glucarpidase is not administered; (2) recommended against extracorporeal treatments when glucarpidase is administered; and (3) recommended against extracorporeal treatments instead of administering glucarpidase. The quality of evidence for these recommendations was very low. Rationales for these recommendations included: (1) extracorporeal treatments mainly remove drugs in the intravascular compartment, whereas methotrexate rapidly distributes into cells; (2) extracorporeal treatments remove folinic acid; (3) in rare cases where fast removal of methotrexate is required, glucarpidase will outperform any extracorporeal treatment; and (4) extracorporeal treatments do not appear to reduce the incidence and magnitude of methotrexate toxicity.

Discovery of Novel Cyclic Ethers with Synergistic Antiplasmodial Activity in Combination with Valinomycin.

With drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4-5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57-65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.

Prospective pan-cancer germline testing using MSK-IMPACT informs clinical translation in 751 patients with pediatric solid tumors.

The spectrum of germline predisposition in pediatric cancer continues to be realized. Here we report 751 solid tumor patients who underwent prospective matched tumor-normal DNA sequencing and downstream clinical use (clinicaltrials.gov NCT01775072). Germline pathogenic and likely pathogenic (P/LP) variants were reported. One or more P/LP variants were found in 18% (138/751) of individuals when including variants in low, moderate, and high penetrance dominant or recessive genes, or 13% (99/751) in moderate and high penetrance dominant genes. 34% of high or moderate penetrance variants were unexpected based on the patient's diagnosis and previous history. 76% of patients with positive results completed a clinical genetics visit, and 21% had at least one relative undergo cascade testing as a result of this testing. Clinical actionability additionally included screening, risk reduction in relatives, reproductive use, and use of targeted therapies. Germline testing should be considered for all children with cancer.

Comprehensive Molecular Profiling of Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1-WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1-WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1-WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study. IMPLICATIONS: These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Novel South African Rare Actinomycete Kribbella speibonae Strain SK5: A Prolific Producer of Hydroxamate Siderophores Including New Dehydroxylated Congeners.

In this paper, we report on the chemistry of the rare South African Actinomycete Kribbella speibonae strain SK5, a prolific producer of hydroxamate siderophores and their congeners. Two new analogues, dehydroxylated desferrioxamines, speibonoxamine 1 and desoxy-desferrioxamine D1 2, have been isolated, together with four known hydroxamates, desferrioxamine D1 3, desferrioxamine B 4, desoxy-nocardamine 5 and nocardamine 6, and a diketopiperazine (DKP) 7. The structures of 1-7 were characterized by the analysis of HRESIMS and 1D and 2D NMR data, as well as by comparison with the relevant literature. Three new dehydroxy desferrioxamine derivatives 8-10 were tentatively identified in the molecular network of K. speibonae strain SK5 extracts, and structures were proposed based on their MS/MS fragmentation patterns. A plausible spb biosynthetic pathway was proposed. To the best of our knowledge, this is the first report of the isolation of desferrioxamines from the actinobacterial genus Kribbella.

Muramyl Tripeptide-Phosphatidyl Ethanolamine Encapsulated in Liposomes (L-MTP-PE) in the Treatment of Osteosarcoma.

The recruitment of autologous macrophages to attack osteosarcoma represents a novel immunotherapy approach to the treatment of osteosarcoma. Muramyl tripeptide-phosphatidyl ethanolamine encapsulated in liposomes (L-MTP-PE) was derived as a compound with the ability to stimulate macrophages to destroy autologous osteosarcoma tumor cells. Preclinical studies including studies in dogs with spontaneously arising osteosarcoma showed the ability of L-MTP-PE to control microscopic metastatic disease in osteosarcoma. A pivotal clinical trial led to the approval of L-MTP-PE for the treatment of newly diagnosed osteosarcoma in over 40 countries.

The clinical heterogeneity of round cell sarcomas with EWSR1/FUS gene fusions: Impact of gene fusion type on clinical features and outcome.

The genetic hallmark of classic Ewing sarcoma is a recurrent fusion between EWSR1 and FUS gene with a member of the ETS transcription factor family. In contrast, tumors with non-ETS gene partners have been designated until recently "Ewing-like sarcoma," as a provisional molecular entity, as their clinical and pathologic features were still evolving. However, this group was reclassified as "round cell sarcoma with EWSR1-non-ETS fusions" in the latest 2020 WHO classification. Moreover, round cell sarcomas with either CIC or BCOR gene abnormalities, initially classified under Ewing family of tumors, are now regarded as stand-alone pathologic entities based on their distinct features. In this study we investigated the clinical characteristics of 226 confirmed Ewing sarcoma patients (EWSR1-FLI1 [n = 176], EWSR1/FUS-ERG [n = 35], EWSR1/FUS-FEV [n = 12], and EWSR1-ETV1/4 [n = 3]) and 14 round cell sarcoma patients with EWSR1-non-ETS fusion (EWSR1/FUS-NFATC2 [n = 10], EWSR1-PATZ1 [n = 3], and EWSR1-VEZF1 [n = 1]). The impact on overall survival (OS) was assessed in 90 patients with available follow-up, treated between 2011 and 2018. Patients with fusions involving FEV and NFATC2 genes showed an older median age at diagnosis, compared to those with EWSR1-FLI1 (P = .005), while extraskeletal location was more common in tumors with noncanonical EWSR1-FLI1 fusions (P = .001). Axial and pelvic primary sites were more common in patients with EWSR1-FLI1 (72%), while tumors with NFATC2 fusions were more frequent in the limb (78%, P = .006). The 3-year OS in patients with EWSR1-FLI1 was 91%, compared to only 60% in patients with alternative fusions (P = .037); the latter group showing a higher rate of metastases at presentation. However, this OS difference was not significant in patients with localized tumor (P = .585). Our study demonstrates significant correlations between fusion subtype and age at presentation, primary tumor sites, and OS, in both conventional Ewing sarcoma and round cell sarcoma with EWSR1-non ETS fusions patients. Larger studies are needed to determine survival differences in localized tumors.