Ewing sarcoma (EwS) is a neoplasm of considerable clinical importance, as it may originate in either bone or soft tissues, and exhibits a high prevalence among adolescents and young adults (AYA). Its therapeutic management requires a multidisciplinary approach combining intensive chemotherapy with local treatment, which may consist of surgical resection, radiotherapy, or a combination of both. With an incidence estimated at approximately 1.5 cases per million in the AYA population, progress in the treatment of this rare malignancy has largely been driven by collaborative clinical trials. These studies have addressed key issues, such as the optimization of chemotherapeutic protocols and the development of more precise risk stratification strategies, thereby enabling the formulation of more individualized therapeutic approaches based on patient-specific characteristics, including the type of genetic fusion present [1] . In this context, patients with localized disease have achieved event-free survival (EFS) rates approaching 75%. However, the treatment regimens employed are not devoid of significant adverse effects, both immediate and long-term, and patients with metastatic or recurrent disease continue to face a markedly unfavorable prognosis. Consequently, there remains a critical need to refine therapeutic interventions and to explore new alternatives that may improve clinical outcomes while simultaneously minimizing the toxicities associated with current treatments [1] [2] .

In 1921, James Ewing first described what is now known as Ewing sarcoma, distinguishing it as a malignant bone tumor separate from osteogenic sarcoma. He noted that the tumor was composed of small round cells, unlike osteosarcoma, which is characterized by large, spindle-shaped cells. Ewing observed that this tumor tended to originate diffusely in the mid-diaphysis of long bones while sparing the epiphyses, leading him to hypothesize an origin in the endothelium of blood vessels within the medullary cavity. Consequently, he named it “diffuse endothelioma of bone” [3] . Aware that osteosarcoma was resistant to radiation, Ewing proposed that this distinct tumor type might be radiosensitive. In 1923, clinical and radiologic similarities between Ewing sarcoma and osteomyelitis were noted, and Ewing observed that many of his patients presented with a febrile syndrome [3] . Subsequently, Coley proposed a possible infectious etiology; however, this hypothesis was later refuted by Pitchard and colleagues, who found no alterations in the host’s immune defense mechanisms. In 1965, Phillips and Higinbotham demonstrated that irradiating the entire diaphysis of the affected bone with doses exceeding 4000 rads reduced local recurrence, a common indicator of metastatic dissemination [4] . Although some surgeons advocated for amputation, patients often developed metastases within one year. The combination of radiotherapy and chemotherapy began to yield promising results, and in 1969, the use of cyclophosphamide led to prolonged disease-free survival [4] [5] . In 1979, S. Weintroub reported a case of avascular necrosis with histological features suggestive of Ewing sarcoma; although only one similar case had been recorded at the time, subsequent studies confirmed that the tumor described by Weintroub was indeed Ewing sarcoma [6] .

Ewing sarcoma is the second most common malignant bone tumor in children, with an incidence rate of approximately three cases per million individuals in the United States. The majority of diagnoses occur during adolescence, with over 50% of cases presenting between the ages of 10 and 20. Fewer than 23% of patients are under the age of 10, and the incidence decreases significantly in adults over 20 years old. This tumor affects males more frequently, accounting for approximately 61% of cases, and occurs almost exclusively in individuals of Caucasian descent, who represent 92% of all cases [2] [7] .

Ewing sarcoma is characterized by a specific genetic translocation that results in the fusion of the EWSR1 gene, located on chromosome 22, with members of the ETS and FET transcription factor families ( Figure 1 ). The FET family (also known as TET) comprises a group of genes encoding proteins with key roles in transcriptional regulation and RNA processing. The acronym FET derives from its three main members:

These proteins share RNA-binding domains and participate in the regulation of gene expression. Among them, EWSR1 is the most frequently involved in gene fusions in Ewing sarcoma. The ETS family (Erythroblast Transformation Specific) consists of over 20 transcription factors that share a conserved DNA-binding domain known as the ETS domain. This domain recognizes specific DNA sequences and regulates the expression of numerous genes involved in:

In Ewing sarcoma, the most commonly involved ETS genes in fusions with EWSR1 include:

These gene fusions (e.g., EWSR1-FLI1 or EWSR1-ERG) result in aberrant fusion proteins that drive tumor proliferation by disrupting normal gene transcription [8] [9] . The most frequent translocation, present in approximately 85% of cases, involves the FLI1 gene on chromosome 11 and produces the t(11;22)(q24;q12) rearrangement. The EWSR1 gene encodes a protein involved in multiple cellular processes, including gene expression regulation and RNA processing. The EWSR1::FLI1 fusion results in a chimeric oncoprotein that functions as an abnormal transcription factor essential for maintaining tumor cell malignancy. Less common fusions include EWSR1::ERG and FUS::ERG. These rearrangements are typically detected using fluorescence in situ hybridization (FISH) ( Figure 2 ) and reverse transcription polymerase chain reaction (RT-PCR). Notably, detection of the EWSR1::ERG fusion by FISH has a higher false-negative rate compared to EWSR1::FLI1 [10] .

Specific chromosomal translocations are frequently identified in human lymphomas and leukemias. The molecular study of these rearrangements has enabled the discovery of novel mechanisms of malignant transformation. Although such translocations are less common in solid tumors, a recurrent t(11;22)(q24;q12) translocation has been described in a group of closely related neoplasms, including osseous Ewing sarcoma, extraskeletal Ewing sarcoma, peripheral neuroepithelioma (PNET), and Askin tumor. Increasing evidence suggests that these tumors share a common origin from the neural crest [6] [9] , albeit with variable degrees of neural differentiation and diverse tissue locations. Collectively, these neoplasms are now referred to as the Ewing sarcoma family of tumors (ESFT). The ESFT includes four major subtypes: extraskeletal Ewing sarcoma, osseous Ewing sarcoma, Askin tumor (localized in the thoracic wall), and peripheral primitive neuroectodermal tumor (PNET). It is important to specify “peripheral PNET” to distinguish it from unrelated tumors originating in the central nervous system. Extraskeletal Ewing sarcoma most commonly occurs in the paravertebral region and lower limbs [6] , while its appearance in the upper extremities is relatively rare ( Table 1 ).

Tumors of the Ewing sarcoma family (ESFT) are characterized by a recurrent cytogenetic alteration involving a translocation between chromosomes 11 and 22. This genetic anomaly is the most common and is detected in approximately 90% of these tumors. The translocation involves the EWS gene, located on the long arm of chromosome 22 (22q12), and the FLI1 gene, situated on chromosome 11q24. The resulting fusion gene, known as EWS-FLI1, is highly specific to the tumor and is present in nearly all cases of ESFT, making it a valuable diagnostic marker. Additional translocations involving EWS have also been identified, although their clinical significance remains unclear. In recent years, several hereditary cancer syndromes have been studied for their potential association with an increased risk of Ewing sarcoma:

The Li-Fraumeni syndrome phenotype can be influenced by various genetic and epigenetic factors. Notably, certain polymorphic variants in the TP53 and MDM2 genes modulate the LFS phenotype. Studies have examined how the TP53 Arg72Pro and MDM2 T309G single nucleotide polymorphisms (SNPs) affect the risk of developing Ewing sarcoma, independent of a clinical diagnosis of LFS. The G allele of the MDM2 T309G SNP was significantly associated with an increased risk of Ewing sarcoma, whereas no relevant association was found with the TP53 Arg72Pro variant. The T309G polymorphism in MDM2 should be prospectively evaluated in Ewing sarcoma patients, regardless of the mutational status of TP53, CHEK2, or POT1 [12] . TP53 analysis remains crucial when exploring germline contributions in Ewing sarcoma. Although current scientific evidence does not establish a clear link between TP53 mutations and Ewing sarcoma risk, assessing deleterious TP53 germline variants in patients and their families may provide the basis for personalized genetic counseling based on familial risk [12] . Current evidence does not establish a direct relationship between pathogenic germline mutations in TP53 and Ewing sarcoma. However, its role remains under investigation, particularly in the context of Li-Fraumeni syndrome. TP53 testing may be considered in selected cases, especially when there is a family history suggestive of hereditary cancer. MDM2: A significant association has been identified between the MDM2 T309G polymorphism and increased susceptibility to Ewing sarcoma, independently of TP53 mutation status. Although these findings are promising, prospective validation is still required before routine clinical implementation can be recommended [12] [13] .

Hereditary retinoblastoma is caused by a germline mutation in the RB1 gene, which not only predisposes to ocular tumors but also significantly increases the risk of developing second primary malignancies. Previous studies have identified recurrent RB1 mutations in Ewing sarcoma cases, prompting investigations into a potential association. Large cohort analyses have shown that individuals carrying RB1 mutations have a higher risk of developing soft tissue sarcomas, even in the absence of prior radiotherapy. Among secondary sarcomas in these patients, leiomyosarcoma is the most frequently observed subtype [12] . While somatic mutations in RB1 have been documented in some cases of Ewing sarcoma, there is no strong evidence supporting a germline predisposition. Therefore, RB1 analysis is not considered part of the standard genetic assessment for this malignancy.

Bloom syndrome is a rare autosomal recessive disorder caused by mutations in the BLM gene (Bloom Syndrome RecQ Like Helicase), which plays a crucial role in genomic stability. The most frequently associated malignancies in Bloom syndrome include leukemias and lymphomas. While the cancer types observed in these patients generally mirror those found in the general population, they tend to occur at significantly younger ages. Although cases of sarcomas have been reported, no significant increase in sarcoma risk has been identified in Bloom syndrome patients [12] . Brohl and colleagues, through sequencing analyses, identified pathogenic or likely pathogenic BLM variants in patients with Ewing sarcoma. While intriguing, these findings warrant further investigation to determine their clinical significance. Given this preliminary evidence, screening for BLM pathogenic variants may be considered in the germline evaluation of patients with Ewing sarcoma, particularly in cases suggestive of genetic predisposition [12] [14] [15] . Pathogenic or likely pathogenic germline variants in BLM have been reported in certain patients with Ewing sarcoma, suggesting a potential predisposing role [14] [15] . Nevertheless, current data are limited, and further studies are necessary to determine its clinical relevance and to justify inclusion in diagnostic protocols.

Step 1. Cell of origin and characteristics

For decades, the cell of origin of Ewing sarcoma (EwS has been a subject of debate. Initially, a neuroectodermal lineage was proposed based on the expression of neuronal markers in tumor cells. However, more recent studies indicate that these neuroectodermal phenotypes are largely induced by the activity of the EWS-FLI1 fusion protein, rather than reflecting a true neural origin [16] . The currently prevailing hypothesis posits that EwS arises from a mesenchymal stem cell (MSC), likely derived from the bone marrow. MSCs exhibit self-renewal capacity and multipotency, allowing differentiation into osteogenic, chondrogenic, and adipogenic lineages. Experimental models have demonstrated that the introduction of EWS-FLI1 into murine or human MSCs can generate cells with a morphology, transcriptomic profile, and epigenetic behavior highly similar to EwS, supporting their role as the cell of origin [17] [18] .

Step 2. Initiating genetic event—t(11;22) translocation

The primary genomic event in EwS pathogenesis is a balanced chromosomal translocation, specifically t(11;22)(q24;q12), resulting in fusion of the EWSR1 and FLI1 genes. This rearrangement occurs in approximately 85% of cases and produces the chimeric oncogenic protein EWS-FLI1, which combines the transcriptional activation domain of EWSR1 with the DNA-binding domain of FLI1, a member of the ETS transcription factor family [19] . In a smaller subset of cases (~10%), similar translocations such as EWS-ERG are observed. These fusions are highly specific and represent a defining molecular hallmark of EwS [16] .

Step 3. Epigenetic reprogramming and differentiation blockade

The oncogenic function of EWS-FLI1 is primarily exerted through epigenetic reprogramming of the host cell genome. EWS-FLI1 binds to GGAA microsatellite repeats and recruits epigenetic co-activators such as p300/CBP, thereby promoting histone acetylation (H3K27ac) and the formation of de novo enhancers [16] [18] . This process activates a network of genes associated with proliferation and maintenance of an undifferentiated state, including IGF1R, MYC, NKX2-2, and EZH2. Simultaneously, EWS-FLI1 represses differentiation genes characteristic of mesenchymal lineages, blocking normal osteogenic and adipogenic pathways. For example, it has been shown to suppress the expression of the cell cycle inhibitor CDKN1A/p21, thereby interfering with physiological cell cycle regulation [20] . This dual mechanism—oncogene activation and differentiation blockade—establishes a highly proliferative, immature, and senescence-resistant phenotype typical of Ewing sarcoma.

Step 4. Cooperating alterations

Although EWS-FLI1 is the central initiating event in EwS tumorigenesis, it is not sufficient to induce full malignant transformation on its own. Multiple studies have demonstrated that additional genetic alterations act cooperatively. Common secondary events include deletion of the CDKN2A gene (~10%), TP53 mutations (~5% - 10%), and functional loss of STAG2 (~15% - 20%), a cohesin complex component essential for chromosomal architecture and segregation [21] . These alterations enhance genetic plasticity and tolerance to replicative stress. Notably, STAG2 loss is associated with more aggressive clinical behavior and increased metastatic potential [16] . These cooperating mutations enable tumor cells to better withstand the transcriptional dysregulation imposed by EWS-FLI1 and to progress toward advanced malignancy.

Step 5. Impact on cell cycle and proliferation

The mitogenic effect of EWS-FLI1 leads to a marked acceleration of the cell cycle. This chimeric protein upregulates genes involved in G1/S and G2/M transitions, including CCND1 (cyclin D1), MYC, and IGF1R, all of which promote cellular proliferation and survival [16] . Concurrently, it represses transcription of negative cell cycle regulators such as CDKN1A/p21 [20] . This dysregulation promotes unchecked proliferation, enabling rapid tumor expansion.

Step 6. Tumor migration and invasion: EMT, CXCR4/CXCL12 signaling, and the role of M2 macrophages

Ewing sarcoma’s ability to invade tissues and metastasize is mediated by a complex interplay of autocrine and paracrine signals within the tumor microenvironment. A key pathway involves the CXCR4 receptor and its ligand CXCL12 (also known as SDF-1). EwS cells express CXCR4, and its activation by CXCL12—highly expressed in tissues such as lung and bone marrow—facilitates directed migration and metastatic seeding [20] . Tumor-associated macrophages (TAMs), predominantly of the M2 phenotype, also play a major pro-metastatic role. These immunosuppressive cells secrete factors such as TGF-β and IL-10 that promote an epithelial-mesenchymal transition (EMT-like program), characterized by loss of intercellular adhesion, cytoskeletal reorganization, and acquisition of motility [22] . In animal models, pharmacologic inhibition of M2 macrophage activity significantly reduces metastatic burden [23] .

Step 7. Immune evasion

Ewing sarcoma exhibits a marked ability to evade host immune surveillance. One of the primary mechanisms is the downregulation or absence of major histocompatibility complex class I (HLA-I) expression, which impairs antigen presentation to cytotoxic CD8+ T cells [20] . Additionally, the tumor expresses immune checkpoint proteins such as PD-L1, HLA-G, and HLA-E, which bind to inhibitory receptors on T cells (e.g., PD-1, NKG2A), inducing T cell anergy and impairing cytotoxic responses. This immunosuppressive environment is further reinforced by high levels of TGF-β secreted by M2 TAMs and tumor cells, which promote the differentiation of regulatory T cells (Tregs) via FOXP3 induction. These Tregs suppress dendritic cell activation and limit the expansion of effector T cells. Collectively, this immunotolerant niche protects the tumor from immune-mediated destruction [20] [22] .

Step 8. Metastatic progression

Metastatic progression remains the most challenging therapeutic hurdle in Ewing sarcoma. Clinically, metastases most frequently involve the lungs, bone, and bone marrow—sites where CXCL12 expression is abundant ( Figure 3 ). Signaling through the CXCR4/CXCL12 axis guides tumor cell homing to these permissive environments [20] . At the molecular level, in addition to chemokine signaling, EWS-FLI1-regulated genes such as CAV1, EZH2, and TNC contribute to extracellular matrix remodeling and increased motility [23] . Loss of STAG2 also plays a dual role, disrupting chromatin organization and promoting a more aggressive metastatic phenotype [21] . These interconnected pathways promote early systemic dissemination and account for the high relapse rates observed in EwS patients.

A population-based study in Sweden, conducted between 1983 and 1995 using data from the Swedish Cancer Registry managed by the National Board of Health in Stockholm, analyzed all cases of osteosarcoma and Ewing sarcoma in patients aged ≤30 years, excluding tumors located in the skull and ribs. The study identified 102 patients with osteosarcoma (61 males and 41 females) and 47 with Ewing sarcoma (28 males and 19 females), revealing a male predominance in both tumor types, with a male-to-female ratio of 1.5:1. The most frequent location was around the knee, where 74% of osteosarcomas (75 cases) and 23% of Ewing sarcomas (11 cases) were found [7] . The most common initial symptom prompting medical consultation was regional pain, either isolated or accompanied by a palpable mass. Specifically, 70% of osteosarcoma patients (71 individuals) and 72% of Ewing sarcoma patients (34 individuals) sought medical attention due to pain alone. An additional 25% of osteosarcoma patients (26 cases) and 15% of Ewing sarcoma patients (7 cases) presented with both pain and a palpable mass. Only 4% of osteosarcoma patients (4 individuals) and 11% of those with Ewing sarcoma (5 individuals) reported no pain at initial consultation, instead presenting with other clinical manifestations [7] . Cases in infants are exceedingly rare. A report from Turkey documented a case in a 6-month-old infant with a femoral tumor, highlighting the diagnostic and therapeutic challenges unique to this population [24] .

Initial Diagnosis

The study by Widhe and Widhe (2000) also reported the frequency of misdiagnoses at initial clinical evaluation for both osteosarcoma and Ewing sarcoma.

Osteosarcoma

The most frequent initial misdiagnosis was tendinitis, accounting for 31% of cases (25 males [41%] and 7 females [17%]). In 12% of patients (12 cases), the physician was unable to provide a definitive diagnosis. Only two patients were initially misdiagnosed with a pathological fracture.

Ewing Sarcoma

Tendinitis was also the most common misdiagnosis in Ewing sarcoma, recorded in 21% of cases (10 patients). An additional 19% (9 cases) presented diagnostic uncertainty. Misdiagnoses were particularly common in tumors located in the pelvis or proximal femur. Among younger patients, the most frequent alternative diagnoses were transient coxitis and osteomyelitis, whereas in older adults, tendinitis and sciatica predominated as erroneous diagnoses [7] .

Staging

Accurate risk stratification in patients with Ewing sarcoma (ES) is essential for therapeutic decision-making and prognostic assessment. Over recent decades, multiple staging systems have been developed, integrating clinical, histological, surgical, and molecular variables.

1.- American Joint Committee on Cancer (AJCC-TNM) Classification

The AJCC-TNM classification is commonly employed to stage EwS and other neoplasms. Its objective is to aid in prognostication by considering tumor size, histologic confirmation, and topographic localization [25] ( Figure 4 ).

2.- European Society for Medical Oncology (ESMO) Classification

The ESMO system categorizes ES into two fundamental groups: localized disease and metastatic disease. This approach prioritizes the presence of metastasis as the most significant survival predictor and guides treatment intensity accordingly. For instance, metastatic patients often receive intensified regimens such as VDC/IE (vincristine, doxorubicin, cyclophosphamide/ifosfamide, etoposide) [16] . A major limitation of this system is its lack of integration of molecular data and histologic response to chemotherapy [26] .

3.- Euro-E.W.I.N.G. 99 Risk Stratification

The Euro-E.W.I.N.G. 99 trial introduced a more nuanced model, dividing patients into standard, high, and very high-risk categories based on tumor volume, anatomical location, and percentage of necrosis after neoadjuvant chemotherapy [27] (Euro-E.W.I.N.G. 99 Trial Investigators, 2012). One strength of this model is its inclusion of tumor necrosis as a prognostic marker, although reliance on imaging for volume estimation may introduce variability.

4.- Rosen Histologic Necrosis Grading (Grades I - III)

This system evaluates histological response to preoperative chemotherapy by estimating the percentage of tumor necrosis:

This grading informs decisions about adjuvant therapy, although intratumoral heterogeneity may underestimate true necrosis, and the system is not applicable to unresectable tumors.

5.- Children’s Oncology Group (COG) Classification

The COG system stratifies patients into low- and high-risk groups using readily assessable clinical factors such as age, tumor size, location, and metastatic status. High-risk patients receive intensified interval-compressed chemotherapy [29] .

6.- Italian Intergroup Staging System

This approach classifies ES based on surgical resectability:

Although useful in surgical planning, this system has been largely superseded by protocols emphasizing neoadjuvant chemotherapy.

7.- Molecular Prognostic Scale

Still under development, this scale incorporates secondary genetic alterations:

8.- MSTS Functional Scale (Musculoskeletal Tumor Society)

This scale assesses postoperative function, including pain, mobility, emotional acceptance, and return to work. Scores > 25 indicate satisfactory recovery, while scores < 20 suggest severe functional limitation [32] . Though subjective, it remains valuable for evaluating quality of life after surgery.

9.- NIH/NCI Scale for Bone Metastasis

This system classifies skeletal metastatic burden into three grades based on extent. While its prognostic value has been validated, it has been partially replaced by metabolic imaging modalities such as PET/CT [33] .

10.- Enneking System for Musculoskeletal Tumors

Traditionally used for musculoskeletal neoplasms, this system distinguishes between low- and high-grade tumors and metastatic status. In ES, all cases are considered high-grade, limiting the system to differentiating between localized (Stage II) and metastatic (Stage III) disease [32] . Its limitation lies in the exclusion of molecular subtypes and treatment response.

Integration of Staging Systems in Clinical Practice

The combination of clinical [27] , histologic (Rosen), and molecular staging systems [31] , allows for more accurate risk stratification and facilitates personalized therapy. For instance, a patient with pelvic metastatic ES, TP53 mutation, and Grade I necrosis would be classified as very high-risk with an adverse genomic profile, thus qualifying for experimental therapies such as PARP inhibitors or immunotherapy [34] . Nevertheless, the lack of international standardization and inequitable access to genomic sequencing technologies remain significant challenges in global ES management ( Table 2 ).

Tumor Locations

Ewing sarcoma most frequently arises in the long bones of the extremities, including the femur, tibia, fibula, humerus, and pelvis. A study involving 976 patients, conducted by the European Intergroup Cooperative Ewing Sarcoma Studies (EI-CESS), reported the distribution of primary tumor sites as follows ( Figure 5 ):

Nevertheless, atypical primary sites have been documented, including the pancreas [35] , uterine cervix [36] , male external genitalia [37] , cervical esophagus [38] , and small intestine [39] . These unusual presentations underscore the importance of considering Ewing sarcoma in the differential diagnosis of extraosseous masses.

Initial Imaging

Conventional radiography remains the first-line imaging modality in the clinical suspicion of a malignant bone lesion. In Ewing sarcoma, plain radiographs frequently reveal a permeative osteolytic lesion in the diaphysis of long bones, accompanied by cortical destruction and characteristic periosteal reactions such as “onion-skin” layering, spiculated patterns, or a Codman triangle—hallmarks of aggressive tumor behavior [16] [40] . Additionally, in approximately 50% of cases, a soft tissue mass may be visualized, evidenced by displacement of fat planes or loss of soft tissue contours ( Figure 6 ). However, radiography has significant limitations in assessing intramedullary tumor extent and involvement of neurovascular structures, thereby restricting its utility in advanced staging [41] . Despite these limitations, its accessibility and rapid execution make it an essential tool for initial evaluation and for guiding further imaging studies.

CT provides high-resolution anatomical detail, particularly useful in assessing cortical destruction, involvement of trabecular bone, and detection of mineralized matrix components ( Figure 7(A) ). Although intralesional calcifications are uncommon in Ewing sarcoma, their identification may aid in differential diagnosis [40] . One of CT’s most relevant applications is in the detection of pulmonary metastases, where its sensitivity surpasses that of radiography or MRI [42] . Additionally, CT is valuable for guiding interventional procedures such as percutaneous biopsies. However, its sensitivity for detecting bone marrow edema or soft tissue infiltration is limited, and ionizing radiation exposure is a concern—particularly in pediatric populations ( Figure 7(B) ) [19] [43] . Consequently, CT is used selectively, mainly for thoracic staging.

MRI is the most accurate imaging modality for evaluating local extension of Ewing sarcoma. On T1-weighted sequences, the lesion typically appears hypoin-tense, reflecting replacement of normal bone marrow. On T2-weighted sequences, it exhibits hyperintensity due to interstitial edema and high tumor cellularity. Following gadolinium contrast administration, heterogeneous enhancement helps differentiate viable tumor areas from necrosis [16] [40] . MRI is essential for assessing soft tissue invasion, neurovascular involvement, and intramedullary spread, playing a central role in surgical planning and response assessment. Despite its high sensitivity, MRI may overestimate tumor volume by including reactive edema or inflammation, a limitation to consider during post-treatment evaluation [41] .

Bone scintigraphy using technetium-99m has historically been used to detect osseous metastases, as it identifies areas of increased osteoblastic activity. However, its specificity is limited, as inflammatory or traumatic lesions can yield false positives [44] . In many centers, it has been supplanted by ^18F-FDG PET/CT, which detects areas of tumor hypermetabolism in both osseous and extrapulmonary sites. PET/CT not only offers superior sensitivity over bone scintigraphy but also provides metabolic data valuable for treatment monitoring and therapeutic decision-making [45] - [47] . Despite its advantages, PET/CT may produce false positives in inflammatory or infectious settings, and is limited by high cost and limited availability.

Clinical Integration of Imaging Techniques

The integration of various imaging modalities offers a comprehensive and stratified assessment of Ewing sarcoma. Radiography serves as the initial diagnostic tool, providing orientation and justification for further studies. MRI is the most accurate modality for delineating anatomical extent and soft tissue involvement, whereas PET/CT stands out as an advanced tool for systemic staging and treatment monitoring [20] [47] . Recent studies have proposed the complementary use of liquid biomarkers—such as circulating tumor DNA (ctDNA)—as a means to detect early relapse and to enhance the monitoring of minimal residual disease [37] . However, factors such as tumor heterogeneity and limited access to high-complexity technologies in resource-limited countries continue to represent substantial challenges for the universal implementation of these strategies [19] .

Biopsy

From a morphological standpoint, Ewing sarcoma consists of sheets of small, round cells with a high nuclear-to-cytoplasmic ratio [48] . It is commonly categorized within the group of “small round blue cell tumors,” which also includes neuroblastoma, alveolar rhabdomyosarcoma, and lymphoblastic lymphoma [49] ( Figure 8 ).

The tumor cells typically present with scant, weakly eosinophilic cytoplasm, often containing glycogen granules that are positive for periodic acid-Schiff (PAS) staining and diastase-sensitive. Nuclei are round, with evenly distributed chromatin and low mitotic activity [15] .

Immunohistochemistry

Immunohistochemical evaluation is essential for distinguishing Ewing sarcoma from other small round cell tumors. Markers such as CD99, NKX2.2, and FLI-1 are fundamental, though their interpretation must always be contextualized alongside molecular studies [50] .

CD99 (MIC2) is the most commonly expressed immunohistochemical marker in Ewing sarcoma, exhibiting strong, diffuse membranous staining in over 95% of cases [10] [51] . Its high sensitivity makes it an excellent screening tool; however, its specificity is limited, as it is also expressed in other small round cell neoplasms, such as T-cell lymphomas, neuroblastomas, synovial sarcomas, and rhabdomyosarcomas [52] [53] ( Figure 9 ). Therefore, it should be interpreted in conjunction with more specific markers and the histological context. In lymphomas, positivity for CD45 or TdT supports exclusion; in rhabdomyosarcomas, coexpression of myogenin and MyoD1 is diagnostically useful [54] [55] .

NKX2.2 is a transcription factor involved in neural differentiation and is directly induced by the oncogenic activity of the EWSR1-FLI1 fusion protein. It exhibits nuclear staining in approximately 90% - 93% of Ewing sarcomas, offering a sensitivity comparable to CD99 but with higher diagnostic specificity [10] [57] . It is typically negative in most other small round cell tumors, including small cell osteosarcoma and small cell lung carcinoma, although weak positivity may be seen in certain neuroendocrine neoplasms, such as desmoplastic small round cell tumors. Therefore, NKX2.2 should be used cautiously and in conjunction with other markers [58] .

FLI-1 is a nuclear marker from the ETS transcription factor family and is expressed as a result of the characteristic t(11;22)(q24;q12) translocation that produces the EWSR1-FLI1 fusion. Nuclear expression of FLI-1 is observed in 70% - 80% of Ewing sarcomas but also in lymphomas, angiosarcomas, and some melanomas, limiting its specificity [53] [55] . Thus, FLI-1 should be interpreted as part of a panel rather than in isolation. Furthermore, in tumors with alternative translocations such as EWSR1-ERG (~10% of cases), FLI-1 may be negative, emphasizing the need for complementary molecular testing [10] [59] .

Negative Markers: Exclusion of Differential Diagnoses

Exclusion of morphologically similar entities is a critical step in the diagnostic algorithm for Ewing sarcoma. For instance:

The absence of these markers, together with positivity for CD99 and NKX2.2, strongly supports the diagnosis of Ewing sarcoma when supported by appropriate histology.

Emerging Markers: PAX7 and EZH2

PAX7 has recently been proposed as a promising nuclear marker for Ewing sarcoma, with reported sensitivity ranging from 85% to 95% and high specificity relative to other small round cell tumors [57] . Its inclusion in immunohistochemical panels has improved diagnostic accuracy, particularly in small or poorly cellular biopsy specimens. Additionally, EZH2—a histone methyltransferase involved in transcriptional repression—has been found to be overexpressed in Ewing sarcoma, in part regulated by EWSR1-FLI1. While its diagnostic utility is still limited, EZH2 expression may hold future prognostic or therapeutic relevance [34] .

Treatment

Historical Context: Euro-E.W.I.N.G. 99

The Euro-E.W.I.N.G. 99 was a landmark international clinical trial designed to improve the treatment and outcomes of patients with Ewing sarcoma, a malignant bone tumor that primarily affects children, adolescents, and young adults. Initiated in 1999, it became one of the largest and most comprehensive studies of its kind. Its primary objective was to evaluate different therapeutic strategies aimed at optimizing survival while minimizing long-term treatment-related sequelae ( Table 3 ) [27] .

Objectives of the Euro-E.W.I.N.G. 99 Study

The trial focused on the following goals:

Study Design

Patients were stratified into risk groups based on tumor size, anatomical location, and the presence of metastases. The three main categories were:

1) Localized Disease (Standard Risk)

Patients with small, localized tumors without evidence of metastasis received standard therapy with chemotherapy, surgery, and/or radiotherapy [49] .

2) Localized High-Risk Disease

Patients with large tumors or those located in anatomically challenging sites (e.g., pelvis) were considered high risk. This group received intensified treatment protocols, including high-dose chemotherapy and autologous stem cell transplantation [26] .

3) Metastatic Disease

Patients with tumors that had spread to the lungs, bone, or other organs received more aggressive treatment approaches, including high-dose chemotherapy and targeted therapies [27] .

Evaluated Treatments

The study investigated several treatment modalities, including:

Key Findings

The Euro-E.W.I.N.G. 99 trial generated several pivotal outcomes:

Impact on Clinical Practice

The Euro-E.W.I.N.G. 99 trial significantly influenced the clinical management of Ewing sarcoma by:

Recent Therapeutic Advances

Since its first description by James Ewing in 1921, Ewing sarcoma (ES) has undergone a profound evolution in therapeutic management. During the early and mid-20th century, the only available treatments were surgery and radiotherapy, which provided limited local control and low cure rates—even in patients with localized disease [62] . It was not until the 1970s that the introduction of chemotherapy significantly improved survival outcomes. Pioneering studies demonstrated that systemic administration of vincristine, actinomycin D, cyclophosphamide, and doxorubicin markedly enhanced overall survival [63] . Subsequently, combination regimens such as VDC/IE (vincristine, doxorubicin, cyclophosphamide/ifosfamide, etoposide) became the standard of care, integrating systemic chemotherapy with local control via surgery or radiotherapy [29] . In the early 21st century, therapeutic efforts focused on intensifying these classical regimens. In the United States, the Children’s Oncology Group (COG) AEWS0031 protocol demonstrated that reducing the interval between chemotherapy cycles from three to two weeks improved event-free survival [29] . In Europe, the Euro-E.W.I.N.G. 99 and Ewing 2008 trials confirmed that consolidation with busulfan and melphalan followed by autologous stem cell transplantation (ASCT) improved outcomes in high-risk patients [42] [63] . Concurrently, national and international clinical guidelines—such as those from the Spanish Sarcoma Research Group (GEIS)—highlighted the importance of a centralized and multidisciplinary approach to managing ES, particularly in patients with poor prognostic features such as inadequate histologic response or large tumor volume [64] . Predictive models for treatment response have refined criteria for therapy intensification. As the molecular biology of ES became better understood, research shifted toward the development of targeted therapies. The main oncogenic driver, the EWS-FLI1 fusion protein, is difficult to inhibit directly, but several associated proteins—such as BAF, CBP/p300, and MLL—have emerged as potential therapeutic targets, particularly through epigenetic modulation [65] . Tyrosine kinase inhibitors have also been explored. Cabozantinib, a dual inhibitor of MET and VEGFR2, demonstrated an objective response rate of 26% in a phase II trial involving patients with advanced ES, positioning it as a potential therapy for refractory disease [66] . The insulin-like growth factor 1 receptor (IGF-1R) pathway, known to promote ES cell proliferation and survival, has been targeted by agents such as ganitumab. However, a phase II trial failed to show improved event-free survival when ganitumab was added to standard chemotherapy [67] . Simultaneously, immunotherapy has gained attention. Immune checkpoint inhibitors such as nivolumab and ipilimumab have shown limited efficacy, though isolated cases have demonstrated prolonged responses [68] . Emerging strategies include CAR-T cells targeting antigens such as GD2, B7-H3, IGF1R, and VEGFR2, as well as bispecific antibodies and tumor vaccines—all in various stages of clinical development [69] [70] .

Recent advancements in CAR-T cell research have introduced fourth-generation constructs with enhanced persistence and safety profiles. Notably, the C7R-GD2.CAR-T cell therapy [71] , and the use of anti-GD2 CAR-T cells in pediatric patients with relapsed/refractory neuroblastoma and other GD2-positive solid tumors [71] , have shown promising preclinical and early clinical outcomes, some of which include patients with Ewing sarcoma. Furthermore, the EGFR806 CAR-T cell trial is evaluating its efficacy in recurrent/refractory solid tumors, including Ewing sarcoma, due to the overexpression of EGFR in a subset of cases [72] . Another relevant study is exploring safety-engineered CAR-T cells specifically targeting sarcomas, representing a significant step toward personalized immunotherapy in bone tumors [73] [74] . While most of these trials remain in early phases (I/II), their translational relevance is growing, especially for high-risk or refractory Ewing sarcoma, where conventional treatment options remain limited.

Ewing sarcoma exhibits a markedly immunosuppressive tumor microenvironment, characterized by a predominance of M2 macrophages, scarce T-cell infiltration, and elevated levels of inhibitory cytokines such as TGF-β and IL-10. The MIF/CD74 axis has recently been identified as a potential immunotherapeutic target to counteract this immunosuppression [75] . On the diagnostic front, detection of minimal residual disease using RT-PCR targeting the EWS-FLI1 fusion transcript has demonstrated greater sensitivity than conventional morphology [62] . Additionally, liquid biopsy techniques employing small extracellular vesicles (sEVs) containing tumor proteins (e.g., CD99, NGFR) and fusion transcripts offer a less invasive and dynamic method for disease monitoring [76] .

In drug repurposing efforts, metformin combined with chemotherapy regimens such as VIT has shown disease control in patients with relapsed ES [77] . Other trials have evaluated the use of the PARP inhibitor talazoparib in combination with temozolomide, yielding partial responses in pediatric patients [78] . Thalidomide has also been reported as an antiangiogenic agent in refractory tumors [79] . At the experimental level, studies have shown that compounds like triamterene induce selective toxicity in cells with DNA repair defects, paving the way for more personalized therapeutic approaches [80] . Additionally, artificial intelligence tools are being applied to predict prognosis and survival by integrating clinical and molecular variables [81] . Given the increasing complexity of ES management, the National Ewing Sarcoma Tumor Board has issued consensus recommendations that include the use of compressed chemotherapy in adults, maintenance therapy, and autologous stem cell transplantation in high-risk settings. The need for international collaborative clinical trials that include patients of all ages is strongly emphasized [82] .

In summary, the evolution of ES treatment reflects a transition from generalist approaches toward increasingly personalized and rational strategies. The integration of targeted therapies, immunomodulation, molecular diagnostics, and predictive computational models heralds a new era in the management of Ewing sarcoma, particularly for patients with advanced or relapsed disease [83] .

The authors thank PAPIIT IN228825 DGAPA, UNAM for partial financial support.