Chromosomal Rearrangements in Ewing Sarcoma

In the mid-1980s the presence of a recurrent reciprocal chromosomal translocation, t(11;22)(q24;q12), was reported in Ewing sarcoma tumors and cell lines. The same translocation was also identified in PNET, supporting the hypothesis that these two tumors represented the same disease exhibiting varying levels of differentiation along a neuronal pathway. Subsequent studies demonstrated that t(11;22)(q24;q12) was present in approximately 83% of Ewing sarcomas.

The early analysis of translocations in Ewing sarcoma demonstrated that the derivative chromosome 22 was always maintained as these tumors and cell lines undergo clonal evolution, whereas the derivative chromosome 11 could be lost. This indicated that the “22;11” derivative is the key chromosomal abnormality, whereas the reciprocal “11;22” is unnecessary. The t(11;22)(q24;q12) is a tumor-specific or somatic mutation because it is not found in the germline DNA of patients.

In addition to the t(11;22)(q24;q12) rearrangement (and related translocations with similar molecular consequences, discussed later), other chromosomal abnormalities have also been reported in Ewing sarcoma. A careful analysis of these sometimes complex karyotypes has revealed a small group of recurrent, nonrandom chromosomal abnormalities. Gains (usually trisomies) of chromosome 8 may be observed in up to 50% of cases, and gains of chromosomes 12 and 1q are seen in approximately 25% of cases. Gains in chromosome 20 have also been reported in 10% to 20% of cases. Interestingly, a chromosomal translocation that is unrelated to the t(11;22)(q24;q12) has been observed in approximately 20% of cases: der(16)t(1;16). This translocation is often found as an unbalanced rearrangement and thus results in partial gains (trisomies and occasionally tetrasomies) of 1q and losses (monosomies) of 16q. The breakpoints of this translocation appear to be variable and range from q11 through q32 on chromosome 1 and from q11.1 through q24 on chromosome 16. This suggests that the translocation does not alter particular target genes at the breakpoint but rather introduces gains of 1q and losses of 16q. Finally, losses at 1p36 have also been observed. More recent high-resolution copy number studies have identified copy number alterations in specific genes or regions of the genome, although the role of specific genomic gains or losses has yet to be determined. There has also been a report of genomic instability, as measured by microsatellite instability and loss of heterozygosity, in Ewing sarcoma. This finding, however, is somewhat controversial, insofar as a second group was not able to replicate the microsatellite instability findings.

The Cloning of EWS/FLI

A major advance in the understanding of the pathogenesis of Ewing sarcoma occurred when the breakpoint of the (11;22)(q24;q12) translocation was cloned in 1992. The translocation breakpoint was localized roughly in the middle of two genes: EWSR1 and FLI1 . EWSR1 had not been previously identified and so was named on the basis of its involvement in the Ewing sarcoma translocation breakpoint ( Ew ing s arcoma r earrangement domain 1 ). FLI1 had been previously identified in mice as the F riend L eukemia Virus i ntegration site. As a result of the translocation event, these two genes become fused and the expressed transcript encodes the EWS/FLI fusion protein ( Fig. 61-3 ).

Fusion of EWS and FLI produces the EWS/FLI protein. Wild-type EWS is characterized by an amino-terminal domain (NTD) , along with three RGG regions and an RNA recognition motif (RRM) that confer RNA-binding properties to the protein. Wild-type FLI contains a pointed domain (PNT) that overlaps with a transcriptional activation domain and an ETS-type DNA-binding domain. The translocation breakpoint regions of each protein are shown. After a translocation event, EWS/FLI is formed. EWS/FLI maintains the NTD of EWS (which functions as both a strong transcriptional activation domain and a transcriptional repression domain in this context), and the DNA-binding domain of FLI.

EWS/FLI and Other TET/ETS Fusions in Ewing Sarcoma

EWS/FLI consists of the amino terminus of EWS fused, in frame, to the carboxyl terminus of FLI (see Fig. 61-3 ). The EWS portion retained in the fusion contains the pseudorepeat domain that harbors a strong transcriptional activation function as well as a transcriptional repression function. The RNA-binding domain of EWS is lost in the fusion protein and instead is replaced by the portion of FLI that contains its ETS DNA-binding domain.

EWS/FLI functions as an oncoprotein. EWS/FLI can induce NIH3T3 cells (an immortalized mouse fibroblast cell line) to exhibit both anchorage independent growth and growth as tumors when injected subcutaneously into immunodeficient mice. Conversely, blockade of EWS/FLI expression or function (via antisense RNA, dominant-negative blocking alleles, or RNA interference) causes Ewing sarcoma cells to lose their transformed phenotype. These data demonstrate that EWS/FLI expression is required for the oncogenic phenotype of Ewing sarcoma. Interestingly, it has been reported that after therapy-induced neural differentiation of Ewing sarcoma, EWS/FLI expression may also be lost. Although these data support an important role for EWS/FLI expression in Ewing sarcoma proliferation and transformation, they do not address whether EWS/FLI formation is the first step in Ewing sarcoma development or whether other oncogenic events occur first and are followed by EWS/FLI formation.

There are three main consequences of the t(11;22)(q24;q12) rearrangement. First, it places the transcriptional regulation of the EWS/FLI fusion under the control of the EWSR1 promoter. This is important because the activity of the FLI1 promoter is limited to hematopoietic (and perhaps neural crest) lineages, whereas the EWSR1 promoter appears to be active in most, if not all, cell types (see previous sections). Second, the translocation results in a loss of one wild-type EWSR1 allele and one wild-type FLI1 allele. Loss of one FLI1 allele is probably of no consequence because wild-type FLI1 is not expressed in Ewing sarcoma. Whether loss of one EWSR1 allele is important in Ewing sarcoma development is not known. The third consequence of the translocation is that it creates the EWS/FLI fusion protein. This is important because although EWS/FLI functions as an oncoprotein, neither wild-type EWS nor wild-type FLI could induce transformation of NIH3T3 cells, thus demonstrating a gain of function for the EWS/FLI fusion. Structure-function analyses of EWS/FLI demonstrated that both the amino terminal EWS domain and the ETS DNA-binding domain of FLI were required for oncogenic transformation of NIH3T3 cells and Ewing sarcoma cells. These results indicate that the fusion protein functions as an aberrant transcription factor. Comparisons of EWS/FLI with wild-type FLI demonstrated that the EWS domain included in the fusion protein could function as a much stronger transcriptional activation domain than the domain of FLI that was lost in the fusion. These results suggested that EWS/FLI induced oncogenic transformation by binding to target genes through its ETS DNA-binding domain and upregulating the expression of those genes through its EWS domain.

The mechanisms by which the EWS portion of the fusion functions as a transcriptional activation domain are only now beginning to be understood. Because wild-type EWS has been shown to interact with TFIID, RNA polymerase II, or the p300/CBP coactivators, it appears likely that these interactions are also important for EWS/FLI function. RNA helicase A (RHA) has also been shown to be important for the transcriptional activity of EWS/FLI. Thus RHA may also function as a coactivator for gene expression mediated by the fusion protein.

More recently, it has been appreciated that EWS/FLI also functions as a transcriptional repressor. For example, analysis of the transcriptional program triggered by EWS/FLI in Ewing sarcoma cells has revealed that more genes are downregulated by the fusion than are upregulated. Additional analysis has demonstrated that EWS/FLI functions as a direct transcriptional repressor at some target genes, like LOX and TGFBR2 . The mechanism of repression appears to be through binding of a transcriptional corepressor complex called NuRD, that includes nucleosome remodeling function, as well as histone deacetylase and histone lysine-specific demethylase activities. Both transcriptional activation and repression functions are required for the oncogenic activity of EWS/FLI in Ewing sarcoma cells.

Analysis of EWS/FLI binding sites in the human genome revealed that the fusion protein binds to sites that contain “classic” ETS-type high-affinity sequences (e.g., ACCGGAAGTG). However, a more interesting and unusual finding was that EWS/FLI was also found to bind to GGAA-containing microsatellite sequences. EWS/FLI binds to these microsatellites and uses them as response elements to activate the expression of target genes, and the transcriptional function is proportional to the length of the microsatellite. This raises the intriguing possibility that polymorphisms in GGAA-microsatellites associated with key EWS/FLI target genes (e.g., NR0B1, CAV1, or GSTM4 ) may alter susceptibility to Ewing sarcoma or perhaps outcome in patients with the disease. It has also been suggested that the lack of GGAA-microsatellites near critical EWS/FLI target genes, such as NR0B1 , in organisms other than humans might be the reason that only humans develop Ewing sarcoma.

In addition to EWS/FLI, other less frequent Ewing sarcoma translocation breakpoints were cloned and revealed a very similar structural organization (see Table 61-1 ). As described, approximately 83% of Ewing sarcoma tumors contain an (11;22)(q24;q12) translocation. Approximately 10% of cases demonstrate an alternate translocation, t(21;22)(q22;q12). Cloning of the breakpoint of this rearrangement revealed that EWS was fused, in frame, to another member of the ETS family, ERG. As in the case of EWS/FLI, the EWS/ERG fusion contains the amino terminus of EWS fused to the carboxyl terminus of ERG, which harbors its ETS DNA-binding domain. Interestingly, the ETS DNA-binding domains of FLI and ERG are 98% identical at the amino acid level. Subsequent investigations demonstrated that EWS/ERG also functioned as an oncoprotein in the NIH3T3 cell model. In subsequent years additional translocations were identified in Ewing sarcoma, including a t(7;22)(p22;q12), t(17;22)(q12;q12), and t(2;22)(q33;q12). Cloning of transcripts from these translocations demonstrated that in each case EWS was fused to another member of the ETS family. Currently, five EWS/ETS fusions have been described (see Table 61-1 ). It is assumed that all EWS/ETS fusion proteins bind similar (if not identical) target genes to induce oncogenic transformation.

Overall, most cases of Ewing sarcoma appear to harbor fusions between EWS and various ETS family members. Rarely, fusions between TLS and ETS family members may be present in Ewing sarcoma instead, as shown by the identification of TLS/ERG fusions in four patients with Ewing sarcoma and the identification of a TLS/FEV fusion in another case (see Table 61-1 ). These findings highlight the similarities across the TET family and across the ETS family. Perhaps the most generic manner of describing the fusion proteins in Ewing sarcoma would be as “TET/ETS fusions.” Because EWS/FLI is the most common fusion identified in Ewing sarcoma, most of the experimental work has been performed using this fusion protein. Experimental findings based on EWS/FLI have only occasionally been confirmed using other Ewing sarcoma fusion proteins.

In addition to variability of fusion partners, variability also exists in the exonic structure of some of the fusion proteins ( Fig. 61-4 ). The breakpoints found in Ewing sarcoma translocations occur in the introns of EWSR1 and FLI1 (and presumably the other ETS factors as well, although in most cases this has not been experimentally confirmed). The breakpoints in the EWSR1 gene are found in one of two regions (3 kb and 1.2 kb in size) present in an approximately 6 kilobase (kb) region that includes four of the gene’s 16 introns. The breakpoints in FLI1 occur over an approximately 50-kb region, which includes six of eight FLI introns. Splicing events join adjacent exons together in the fusion transcript. As a result of this variability in breakpoint and subsequent RNA splicing, at least 10 different EWS/FLI isoforms are generated (see Fig. 61-4 ).

Translocation breakpoints may occur within one of two portions of a 6-kb region of the EWSR1 gene and in a 50-kb region of the FLI1 gene. This variety results in the observed heterogeneity of EWS/FLI fusion transcripts, as indicated.

The most common Ewing sarcoma translocation fuses exon 7 of EWSR1 to exon 6 of FLI1 . This “7/6” fusion is commonly referred to as a “type 1” EWS/FLI fusion and accounts for approximately 50% to 60% of EWS/FLI fusions. A “type 2” fusion joins exon 7 of EWSR1 to exon 5 of FLI1 and is the second most common EWS/FLI fusion, accounting for approximately 20% to 30% of EWS/FLI fusions. Laboratory studies suggested that the type 1 EWS/FLI fusion was a weaker oncoprotein than the type 2 fusion, and originally it was thought to have prognostic significance. However, these functional differences were not reflected on changes in gene expression between type 1 and type 2 fusions, and large-scale clinical correlative data did not indicate a prognostic difference based on fusion subtype.

Although there is general agreement regarding the designation of the type 1 and type 2 fusions, there is no generalized nomenclature for the remaining fusions, and so these are best described in terms of which exons are fused to one another. In addition to variability in the EWS/FLI breakpoint, EWS/ERG has also been reported to demonstrate variability based on fusion points, with at least four fusions reported. Because the other fusions are so rare, there is only minimal information as to whether they also show similar levels of breakpoint heterogeneity.

Ewing Sarcoma Cell of Origin

The cell of origin of Ewing sarcoma is unknown, and the subject has generated intense debate. The original description by James Ewing in 1921 suggested that this “round cell sarcoma” was of endothelial origin and hence properly referred to as a “diffuse endothelioma of bone.” Dr. Ewing later suggested that it may arise from perivascular lymphatic endothelium. A hematologic origin was proposed in the early 1970s following an in-depth evaluation of light and electron microscopic characteristics and cytochemistry of both primary tumor and cell culture specimens. In the early 1980s a fibroblastic-mesenchymal derivation was proposed based on, among other things, the patterns of collagen expression of the tumor. In the late 1980s, however, a neural crest derivation was proposed because of the occasional identification of certain neural features, such as the presence of Homer Wright rosettes, neural processes, neurosecretory granules, and neural immunohistochemical markers. Additionally, undifferentiated Ewing sarcoma cell lines can occasionally be induced to express markers of neural differentiation, including the expression of neuritelike elongated processes, neuron-specific enolase (NSE), cholinesterase, and neural filament triplet protein. Interestingly, there are a few examples of neural differentiation of Ewing sarcoma occurring after therapy, which further supports this theory. More recently, it was shown that introduction of EWS/FLI into murine mesenchymal stem cells could result in oncogenic transformation, suggesting that these may be the cell of origin (although the same does not hold true for human mesenchymal stem cells). This hypothesis was further supported by microarray and cell biological studies performed on Ewing sarcoma cell lines in which EWS/FLI expression was reduced using RNA interference techniques. In the absence of EWS/FLI expression, these cells had a gene expression pattern that was reminiscent of mesenchymal stem cells and a cellular structural organization and adhesive and migratory properties that were also consistent with a mesenchymal phenotype. Furthermore, Ewing sarcoma cells with reduced EWS/FLI expression could be induced to differentiate into other mesenchymal cell types, such as fat and bone. This is a characteristic that is shared by mesenchymal stem cells.

It is important to note, however, that the phenotype of Ewing sarcoma (at least the neural crest component) may be a consequence of the (11;22) translocation rather than being related to the tumor’s cell of origin. Introduction of EWS/FLI into NIH3T3 murine fibroblasts induces a phenotype that is highly similar to Ewing sarcoma, including the small round blue cell morphology and features of neural differentiation. Similarly, expression of EWS/FLI in human rhabdomyosarcoma cells, neuroblastoma cells, or even normal human fibroblasts causes those cells to express genes that are similar to the genes expressed in Ewing sarcoma. This suggests that EWS/FLI may cause transdifferentiation of its target cells. Thus the neural crest phenotype of Ewing sarcoma may be induced by EWS/FLI.

Transcriptional profiling experiments (e.g., those using oligonucleotide or complementary DNA microarrays and, more recently, RNA sequencing) in Ewing sarcoma tumors and cell lines have allowed for a comprehensive analysis of the gene expression patterns associated with this disease. One of the earliest studies compared Ewing sarcoma tumors and cell lines with other small round cell tumors of childhood, including neuroblastoma, non-Hodgkin lymphoma, and rhabdomyosarcoma. These studies demonstrated that these histologically similar tumors could be distinguished from one another using gene expression data. A more recent analysis compared the expression of approximately 180 sarcoma tumor samples and again showed that tumors could be distinguished using gene expression patterns. Additional studies in which the gene expression pattern of Ewing sarcoma was compared with that of a variety of normal tissues supported the neural crest derivation but also suggested some similarity to endothelial cells. As discussed in the preceding section, analysis of some model systems has suggested a mesenchymal stem cell origin. A definitive understanding of the cell of origin of these tumors will require additional studies. It should be noted that as of yet no genetically engineered mouse model of Ewing sarcoma has been reported, and therefore tissue-specific oncogene expression or lineage-tracing approaches have not been used to identify the Ewing sarcoma cell of origin.

EWS/FLI Target Genes

Because most studies have suggested that EWS/FLI functions as a transcription factor, the two biggest questions in the field have been (1) what genes are dysregulated by EWS/FLI and (2) what are the roles of these gene targets in oncogenic transformation? These questions are beginning to be answered. Because the cell of origin of Ewing sarcoma is unknown, most laboratory-based studies of EWS/FLI have used heterologous cell types with varying results. More recent approaches have benefited from the use of “knockdown” (reduction) of gene expression using RNA interference (RNAi). The RNAi technique has allowed for the analysis of EWS/FLI in patient-derived Ewing sarcoma cell lines themselves, thus avoiding the concerns associated with using heterologous cell types.

Gene expression analysis of Ewing sarcoma cells in which EWS/FLI has been knocked down has allowed a comprehensive identification of the thousands of genes that are modulated by the fusion protein. For example, increased expression of NKX2.2 and NR0B1 are required for the oncogenic phenotype of Ewing sarcoma, although the specific mechanisms by which these transcriptional regulators facilitate the transformed phenotype remains poorly understood. Similarly, repression of TGFBR2 (the receptor for transforming growth factor β) and lysyl oxidase (LOX) by EWS/FLI are also required for tumorigenicity of Ewing sarcoma cells.

Gene expression studies also demonstrated that EWS/FLI represses the expression of the insulin-like growth factor binding protein 3 (IGFBP-3). IGFBP-3 appears to be involved in Ewing sarcoma apoptosis, and thus decreased expression of this protein appears to prevent apoptosis. These data complement a growing body of literature that supports a critical role for IGF-1 signaling in Ewing sarcoma development. For example, it was shown that Ewing sarcoma tumors and cell lines express IGF-1 and IGF receptors. IGF-1 and its receptor appear to function in an autocrine/paracrine fashion in Ewing sarcoma, and disruption of this signaling pathway blocks various functions that contribute to tumorigenesis. This occurs both in Ewing sarcoma cell lines as well as in model systems. Indeed, disruption of this pathway has shown some efficacy as a molecularly targeted therapeutic approach for this disease (discussed later).

NPY1R was identified as an EWS/FLI target gene through microarray analysis. Neuropeptide Y (NPY) is a small polypeptide neurotransmitter with diverse functions. Engagement of NPY receptors by NPY in Ewing sarcoma cell lines is growth inhibitory, suggesting a potential therapeutic role for these receptors. This result was surprising, insofar as Ewing sarcoma cells also express the NPY ligand. It appears likely that Ewing sarcomas are protected from the growth-inhibitory effect of NPY/NPY1R interactions by the expression of dipeptidyl peptidase IV, which cleaves NPY into a form that is unable to bind NPY1R. This cleaved form, NPY _3-36 , functions as an angiogenic factor by binding to NPY2R receptors on endothelial cells, thus providing a likely explanation for the maintenance of this growth-inhibitory autocrine pathway in Ewing sarcoma cells.

Another protein that has been shown to be upregulated by EWS/FLI is vascular endothelial growth factor (VEGF). VEGF levels are increased in Ewing sarcoma cell lines and xenografts. VEGF levels are also increased in the blood of patients with Ewing sarcoma and VEGF has been shown to be expressed in 55% of Ewing sarcoma tumor samples by immunohistochemistry. In the case of Ewing sarcoma, VEGF may induce not only angiogenesis but also vasculogenesis. Blockade of VEGF function in Ewing sarcoma model systems prevents tumor growth. These preclinical studies demonstrate the potential for VEGF inhibition as a therapeutic strategy for Ewing sarcoma.

Most, but not all, studies of Ewing sarcoma have shown that these tumors typically express telomerase activity. Interestingly, it appears that hTERT, the catalytic subunit of the telomerase holoenzyme, is upregulated by EWS/ETS proteins. This upregulation appears to be caused by specific binding of the ETS portion of the fusion to the TERT promoter. These data suggest that EWS/ETS fusions regulate telomerase activity by direct activation of the TERT promoter.

One key protein whose expression may be regulated by EWS/FLI is CD99 (also called MIC2). In human fibroblasts engineered to express EWS/FLI, the expression of CD99 closely mimicked that of the fusion protein. In the early 1990s it was found that CD99 is expressed at high levels in Ewing sarcoma cells but is uncommonly found on other tumor types. The CD99 antigen is recognized by a variety of monoclonal antibodies, including 12E7, HBA71, and O13, and these antibodies have an important role in the diagnosis of Ewing sarcoma (discussed later). The CD99 antigen is a membrane glycoprotein that plays a role in T-cell development and activation and migration of monocytes through endothelial junctions. In Ewing sarcoma CD99 inhibits neural differentiation and thus contributes to oncogenic transformation. Significantly, emerging data suggest that antibody-mediated engagement of CD99 may eventually be exploited as a new therapeutic approach to Ewing sarcoma. CD99 engagement in Ewing sarcoma results in apoptosis of the tumor cells. This effect enhances tumor cell killing by chemotherapeutic agents. Gene expression studies of CD99-mediated apoptosis suggested a role for the cytoskeletal protein zyxin in this process, and this finding was confirmed through functional studies. The identification of zyxin in this process was interesting in light of subsequent studies demonstrating that zyxin acts as a tumor suppressor in Ewing sarcoma. Taken together, these studies suggest that CD99 may be an effective therapeutic target in Ewing sarcoma by modulating cytoskeletal actin function.

Cooperative Pathways in Ewing Sarcoma Oncogenesis

Multiple potentially cooperating mutations in addition to TET/ETS fusions have been identified in Ewing sarcoma. The most widely assessed are mutations in the p53 and RB pathways. The p53 pathway includes proteins such as p14 ^ARF and MDM2, as well as p53 itself. When expressed in some heterologous systems, such as primary human fibroblasts, EWS/FLI induces the expression of p53. This finding suggests that there is selective pressure to inhibit the p53 pathway. This hypothesis appears to be at least partially true. Deletions in the CDKN2A locus (commonly called the INK4a locus encoding both the p14 ^ARF and p16 ^INK4a proteins; note that p14 ^ARF is involved in the p53 pathway, whereas p16 ^INK4a is involved in the RB pathway) have been identified in approximately 10% to 30% of primary Ewing sarcoma tumors and more than 50% of Ewing sarcoma cell lines. Amplifications or overexpression of MDM2 is occasionally seen in Ewing sarcoma. Mutations in p53 are found in 5% to 20% of Ewing sarcomas. Taken together, mutations in the p53 pathway may be present in nearly 70% of Ewing sarcomas, and these mutations may have prognostic significance in this disease (discussed later). Because there are likely additional components of the pathway yet to be identified or analyzed, the frequency of alterations in this pathway may be even higher.

The RB pathway regulates the cell cycle and includes proteins such as CDK4, D-type cyclins, and p16 ^INK4a . Mutations have been found in RB itself, and deletions of the INK4a locus have been documented as well in Ewing sarcoma. As was observed in the case of the p53 pathway, mutations in the RB pathway also appear to be relatively common in this disease.

Whereas mutations in the p53 and RB pathways are relatively common in Ewing sarcoma, mutations in other genes or pathways have only rarely been identified. For example, mutations in BRAF (which is also a member of the RAS/MAPK pathway) have been identified in 1 of 22 cell lines examined. No mutations in other components of the pathway, including RAS or receptor tyrosine kinases, have been identified, as of yet. This suggests that the RAS/MAPK pathway is only rarely activated by mutation in this disease.

Although the RAS/MAPK pathway does not appear to be frequently activated by mutation, there is some evidence for a requirement of pathway activation in Ewing sarcoma tumor development. In the NIH3T3 model system, it was shown that expression of EWS/FLI induced phosphorylation of the ERK1 and ERK2 proteins (downstream members of the MAPK pathway). This effect may result from autocrine stimulation of these transformed cells by PDGF-C, which was also shown to be induced by EWS/FLI expression in the NIH3T3 model, and expressed in Ewing sarcoma cell lines and tumor specimens. There is some controversy as to whether PDGF-C plays a role in the human disease, as PDGFR-α, which is required for PDGF-C responsiveness, is not expressed in Ewing sarcoma. Although PDGF-C may not be involved in Ewing sarcoma, inhibition of the MAPK pathway does block oncogenic transformation mediated by EWS/FLI in the NIH3T3 model. This finding was not replicated in Ewing sarcoma cells, thus raising the question as to whether this is an NIH3T3-specific pathway.

Many members of the Wnt signaling pathway are expressed in Ewing sarcoma, including soluble Wnt ligands, Wnt receptors (frizzled), and Wnt coreceptors (LRP5/6). Additionally, microarray analysis of EWS/FLI target genes has demonstrated downregulation of the Wnt inhibitor DKK1 and upregulation of various Wnt signaling components. These data suggest that Wnt signaling occurs in Ewing sarcoma. In contrast, analysis of the pathway in Ewing sarcoma cell lines in tissue culture has not demonstrated evidence of active Wnt autocrine signaling. If the pathway is involved in Ewing sarcoma, it seems likely that it is important for processes such as migration and metastasis, rather than proliferation and transformation. Current studies have not addressed this possibility.

Finally, genes involved in chromosomal stability or chromatin remodeling have also been implicated in Ewing sarcoma. For example, the STAG2 protein helps regulate chromosomal separation during mitosis and was found to be deleted in a number of human tumor types, including Ewing sarcoma. It was suggested that this may be a mechanism of chromosomal instability in Ewing sarcoma and other tumors. Deletion of SMARCB1 (encoding the SMARCB1/INI1/SNF5 protein) was found in approximately 10% of Ewing sarcoma cases. SMARCB1 is a member of the SWI/SNF chromosome remodeling complex whose deletion is tightly associated with atypical teratoid/rhabdoid tumor. Whether SMARCB1-deleted Ewing sarcoma tumors represent a unique subset of Ewing sarcoma or instead indicate that alterations in chromatin remodeling complexes are widespread in Ewing sarcoma is currently unknown. Interestingly, a newly identified chromosomal rearrangement, t(4;22)(q31;q12), encodes an EWS/SMARCA5 fusion protein in tumors that appear consistent with Ewing sarcoma, supporting the idea that alterations in this chromatin remodeling complex may indeed be widespread in this disease. Another possibility is that the EWS/SMARCA5 fusions are found in tumors that are similar, but not identical, to Ewing sarcoma. Indeed, the most recent World Health Organization guidelines have named these (and other non-TET/ETS translocation-bearing tumors that have similar appearance to Ewing sarcoma; see Table 61-1 ) as Ewing-like sarcomas.

Apoptotic Pathways in Ewing Sarcoma

As described previously, mutations in the p53 pathway are relatively common in Ewing sarcoma. These mutations likely confer resistance to the growth effects of TET/ETS fusions in the disease and may also protect against apoptosis. Whereas p53 is an important mediator of apoptosis, other signaling pathways are involved as well. These other pathways have been evaluated in Ewing sarcoma in a number of studies.

Both Fas and the Fas ligand (FasL) are expressed in the majority of Ewing sarcomas. The status of the FasL in Ewing sarcoma is somewhat controversial; one study reported soluble FasL in Ewing sarcoma–conditioned media, whereas another study reported that the FasL is only found intracellularly and thus is not accessible to cell surface receptor. In terms of the receptor, Fas at the cell surface can be functional, but Ewing sarcoma cells can be divided into Fas-sensitive, Fas-inducible, and Fas-resistant groups. Fas-sensitive lines are killed by coincubation with a FasL-expressing effector cell. Fas-inducible lines are killed by this effector cell line only after pretreatment with interferon-γ (IFN-γ) or cycloheximide (or both). The Fas-resistant lines were resistant regardless of pretreatment. The differences between sensitive and resistant cells are not completely known but may involve differences in Fas expression or expression of proapoptotic or antiapoptotic mediators, such as BAD and BAR. Strategies to improve Fas-mediated killing of Ewing sarcoma cells have been investigated, such as allowing for accumulation of more FasL through the use of metalloproteinase inhibitors and upregulation of Fas by administration of interleukin-12.

In addition to Fas/FasL, evaluations of tumor necrosis factor–related apoptosis-inducing ligand and tumor necrosis factor α as inducers of Ewing sarcoma apoptosis have also been conducted, in vitro and in vivo, alone and in combination with other agents. Taken together, these data support a potential use of proapoptotic ligands in the treatment of Ewing sarcoma. However, the variability of the effects seen demonstrate the need for an ongoing mechanistic evaluation of these pathways in this disease.

Biological Features Summary

The identification of t(11;22)(q24;q12) and subsequent cloning of the EWS/FLI fusion protein represented a major advance in the understanding of the pathogenesis of Ewing sarcoma. EWS/FLI appears to function primarily as an aberrant transcription factor to dysregulate gene targets involved in the development of this disease. Multiple gene targets and cooperating molecular pathways have been identified. These represent a wide array of functions, including growth factor signaling, survival and apoptosis pathways, angiogenesis, cellular immortalization, and cellular differentiation. Despite these myriad molecular functions, a unified mechanistic understanding of Ewing sarcoma development has not yet been reached. Similarly, the cell of origin of the disease has not yet been clearly identified. Nonetheless, there is great hope that a detailed understanding of the molecular mechanisms involved in Ewing sarcoma development will lead to new diagnostic, prognostic, and therapeutic approaches for this disease.

Clinical Presentation

The most common presenting symptoms of Ewing sarcoma are pain, a palpable mass, or both. In one series 89% of patients with Ewing sarcoma reported pain at the time of initial presentation. Pain does not necessarily occur at night; this pattern was reported in only 19% of patients in one series. Patients may report that their pain began at the same time as a minor musculoskeletal injury. This presentation, reported in approximately 25% of patients, may delay the diagnosis. Unlike patients with osteosarcoma, patients with Ewing sarcoma may report constitutional symptoms such as fever and weight loss. Approximately 15% to 20% of patients have fever at presentation. Other symptoms depend on the site of disease. For example, 40% to 94% of patients with paraspinal tumors have presenting symptoms of spinal cord compression. Patients with large pelvic tumors may complain of an alteration in voiding. Extensive bone marrow metastatic disease may cause symptoms related to anemia, thrombocytopenia, or neutropenia. Patients may have symptoms for many weeks or months before seeing a physician for evaluation, with most studies reporting an average time from symptom onset to diagnosis of 3 to 5 months.

Ewing sarcoma can arise from any bone but has a predilection for pelvic bones and long bones of the leg. Approximately 45% of Ewing sarcomas develop in the axial skeleton. More than half of these axial tumors arise from pelvic bones, and a quarter of axial tumors arise from a rib. Up to 30% of Ewing sarcomas arise from long bones in the leg, and up to 15% of tumors develop in long bones in the arm. Tumors of the hands, feet, and head develop only rarely, accounting for 10% or fewer of all tumors. The distribution of primary tumor site does not vary among racial groups. Soft tissue tumors may arise in any location, although axial sites predominate.

Laboratory studies in patients with newly diagnosed Ewing sarcoma may reveal nonspecific abnormalities. More than a third of patients have an elevated erythrocyte sedimentation rate (ESR) at the time of diagnosis. Similarly, serum lactate dehydrogenase (LDH) levels are elevated in approximately one third of patients. Laboratory manifestations of tumor lysis syndrome are uncommon with these tumors. Unlike osteosarcoma, alkaline phosphatase is not typically elevated in patients with Ewing sarcoma of the bone. Patients with advanced bone marrow metastatic disease may have anemia, thrombocytopenia, and neutropenia detected on a complete blood count. In one large series 12% of patients had anemia even in the absence of metastatic disease.

Box 61-1 summarizes the differential diagnosis of Ewing sarcoma. In most cases of Ewing sarcoma of the bone, the main alternative diagnosis is osteosarcoma. Table 61-2 displays features that may help differentiate Ewing sarcoma of the bone from osteosarcoma. Compared with osteosarcoma, Ewing sarcoma is more likely to occur in the axial skeleton. Those Ewing tumors that arise in long bones have a greater tendency to occur in the diaphysis, compared with the tendency of osteosarcoma to arise in the metaphysis of long bones. Patients with Ewing sarcoma are more likely to report constitutional symptoms than are patients with osteosarcoma. Other malignant tumors in the differential diagnosis of Ewing sarcoma of the bone include primary bone lymphoma, giant cell tumor of the bone, and bone metastasis of an extraosseous malignancy. The differential diagnosis of Ewing sarcoma arising from the soft tissues is more broad because the site of origin for these tumors is not restricted to the bone. The site of origin may help narrow the differential diagnosis. Paraspinal tumors may be confused with neuroblastoma, particularly in younger patients. Pelvic tumors may suggest rhabdomyosarcoma or malignant germ cell tumor. Lymphoma and other soft tissue sarcomas can arise at any site and should be included in the differential diagnosis.

Reports from several cooperative group studies have defined the metastatic behavior of Ewing sarcoma. Approximately 25% of patients exhibit distant metastases at presentation. Rates of metastatic disease do not appear to differ between patients with skeletal tumors and those with soft tissue primary tumors. Patients with pelvic primary tumors have an increased incidence of metastatic disease at initial diagnosis. The lung is the most common metastatic site, with pulmonary dissemination reported in 50% to 60% of patients with metastatic disease. Patients with isolated pulmonary metastasis represent between 26% and 36% of cases of metastatic disease. Patients usually have multiple lung nodules identified in both lungs.

The bone is the next most common metastatic site at presentation. One group reported cortical bone metastases in 43% of patients with metastatic disease. A large cooperative group reported that the bone marrow is involved in approximately 19% of metastatic patients at initial diagnosis. A smaller series noted bone marrow involvement in 52% of patients with metastatic disease. This group performed up to 10 bone marrow aspirates on patients at initial presentation, suggesting that routine sampling may underestimate the incidence of bone marrow involvement. Brain metastasis at initial diagnosis appears to be extremely uncommon. In two series of patients with brain metastasis from Ewing sarcoma, all cases were reported at the time of disease recurrence and not at initial presentation.

Fewer than 10% of patients have lymph node involvement at initial diagnosis. The incidence of node involvement is higher in patients with soft tissue rather than bone primary tumors. In one population-based study, the incidence of regional node involvement among patients with soft tissue tumors was 12.4% compared with 3.2% in patients with bone primary tumors. The incidence of regional node involvement is also higher among patients with distant metastatic disease.

This metastatic pattern helps determine the appropriate staging evaluation for patients with Ewing sarcoma ( Box 61-2 ). In addition to computed tomography (CT) or magnetic resonance imaging (MRI) scans of the primary tumor, all newly diagnosed patients should be evaluated with a chest CT scan. Assessment for bone metastasis has conventionally been with radiolabeled-technetium bone scan. More recent data indicate that positron emission tomography (PET) scans are superior for this indication (discussed later), prompting wider adoption of PET imaging at initial diagnosis. At least bilateral bone marrow aspirates and biopsies should routinely be performed in newly diagnosed patients, with biopsy more sensitive for detection of marrow involvement compared with aspirate. Attention to regional nodes on imaging studies is particularly warranted in patients with soft tissue primary tumors. These evaluations will indicate whether a patient has metastatic or nonmetastatic Ewing sarcoma at initial diagnosis. This distinction serves as the main staging classification in practice for these patients. Although a staging classification for musculoskeletal tumors has been devised by Enneking, this system is not routinely used in the clinical care of patients with Ewing sarcoma.

Imaging Features

Most patients with Ewing sarcoma of the bone are initially evaluated with a plain radiograph. Two representative radiographs are shown in Figure 61-5 . These tumors typically appear as poorly circumscribed lesions arising from the bone, but with an associated soft tissue mass. Both lytic and sclerotic areas may be seen within the bony component of the tumor. Given the aggressive nature of these tumors, periosteal reaction is commonly observed. A layered or laminated periosteal reaction is most typical, often giving an onion-skin appearance (see Fig. 61-5, A ). A spiculated “sunburst” periosteal reaction is more commonly associated with osteosarcoma but may be occasionally observed in Ewing sarcoma (see Fig. 61-5, B ). Codman triangle, a triangular area forming under the periosteum in the setting of a rapidly growing bone lesion, may also be seen in some cases. Cortical thickening is present in approximately 20% of tumors. Pathologic fractures occur in 15% of patients with Ewing sarcoma of bone.

A , Plain radiograph of a femoral Ewing sarcoma demonstrating a laminated periosteal reaction, resulting in an “onion-skin” appearance. B , Plain radiograph of a tibial Ewing sarcoma with a spiculated periosteal reaction.

CT scans and MRI scans are used to better define the extent of local tumor in patients with both osseous and extraosseous tumors. CT scans most commonly demonstrate a heterogeneous mass with heterogeneous contrast enhancement. The soft tissue component generally has lower attenuation than muscle on non-enhanced CT scans. MRI scans typically reveal a mass that is heterogeneous with respect to both signal intensity and gadolinium contrast enhancement. Typically T1- and T2-weighted MRI scans demonstrate areas of increased signal intensity of the soft tissue component compared with skeletal muscle. Although both CT and MRI are able to delineate the soft tissue component of the tumor, MRI may be better able to determine the extent of bone marrow extension and the degree of growth plate involvement.

Emerging evidence suggests that fluorodeoxyglucose PET (FDG-PET) may play an increasing role in the management of patients with Ewing sarcoma. In one series all 32 patients with Ewing sarcoma evaluated by FDG-PET imaging before initiation of chemotherapy had FDG-avid tumors. Figure 61-6 demonstrates an FDG-PET scan of a patient with widely metastatic Ewing sarcoma at initial presentation. A group of studies has evaluated the role of FDG-PET imaging in screening for metastatic disease and for disease recurrence. Several groups have demonstrated that FDG-PET imaging has superior sensitivity and specificity compared with conventional bone scans for detecting bone metastases in patients with Ewing sarcoma. In contrast, FDG-PET appears to be inferior to spiral CT scans in screening for pulmonary metastases in these patients. In screening patients with Ewing sarcoma for overall disease recurrence at any site, FDG-PET imaging may have lower sensitivity but higher specificity compared with conventional imaging. Finally, one group has reported that a decrease in FDG-avidity in response to neoadjuvant chemotherapy correlates with improved progression-free survival.

Whole-body fluorodeoxyglucose positron emission tomography (FDG-PET) scan of a patient with widely metastatic Ewing sarcoma at initial diagnosis. FDG uptake is evident in bones, soft tissue, and bone marrow.

Pathologic Diagnosis

Once imaging studies have localized the tumor, diagnostic tissue must be obtained. The current pathologic diagnosis of Ewing sarcoma depends on tumor morphology, immunohistochemistry findings, and demonstration of a Ewing sarcoma–specific translocation (by standard cytogenetics, fluorescence in situ hybridization [FISH], or reverse transcriptase polymerase chain reaction assays [RT-PCR]). In planning for biopsy, the physician must ensure that adequate tissue will be available to perform each of these tests. For example, one series reported successful cytogenetic analysis in only 59% of fine needle aspirations performed for Ewing sarcoma. The biopsy should be planned with an eye toward ultimate local control of the tumor. If surgical resection is ultimately planned, the biopsy tract should be included in the resected specimen.

Ewing sarcoma and PNET form a morphologic continuum ranging from tumors with no apparent neural differentiation to tumors with evidence of early neural differentiation. Of note, the histologic classification of these tumors does not imply site of origin because tumors with either histology may arise in the bone or soft tissue. Ewing sarcoma appears as relatively monomorphic small round blue-staining cells with scant cytoplasm ( Fig. 61-7 ). The nuclei tend to be round without prominent nucleoli. These cells demonstrate positive periodic acid–Schiff staining owing to cytoplasmic glycogen. Electron microscopy may reveal these glycogen stores. The tumor cells grow in sheets without additional structure. A large cell or atypical Ewing sarcoma subset has been described with large irregular nuclei and more conspicuous nucleoli. As many as 30% of tumors with Ewing sarcoma histology arise in soft tissues rather than bone. PNETs typically display primitive Homer Wright pseudorosette formation. Electron microscopy may reveal additional features suggestive of neural differentiation, such as neurosecretory granules. Tumors without pseudorosette formation may also be classified as PNETs if they express two or more neural markers (discussed later). Approximately 50% of tumors with PNET histology arise in bone. Despite these morphologic differences, Ewing sarcoma and PNET are now viewed as the same biological entity.

A , Characteristic morphology of Ewing sarcoma with monomorphic small round blue cells without pseudorosette formation. B , Morphologic features of a primitive neuroectodermal tumor (PNET), including a pseudorosette formation in the center of the panel. C , The characteristic strong membranous CD99 immunohistochemical staining of Ewing sarcoma.

(Courtesy Dr. Antonio Perez-Atayde, Department of Pathology, Children’s Hospital, Boston, Mass.)

Immunohistochemistry plays a key role in differentiating Ewing sarcoma and PNET from other small round cell tumors of children and young adults. The most useful antigen in the diagnosis of Ewing sarcoma appears to be CD99. This protein product of the MIC2 gene is expressed in a limited number of normal tissues, including strong staining of ependymal cells, pancreatic islet cells, anterior pituitary gland, testicular Sertoli cells, ovarian granulosa cells, and maturing T lymphocytes. Less intense staining has been noted in scattered endothelial cells. Ewing sarcoma and PNET both display prominent CD99 immunostaining in a membranous pattern (see Fig. 61-7 ). Multiple series have found strong CD99 immunostaining in more than 90% of cases of Ewing sarcoma and PNET. Staining for neural markers, such as synaptophysin, S-100, NSE, and neurofilament, is variable and helps to characterize tumors as Ewing sarcoma or PNET. These tumors also commonly stain with vimentin, whereas desmin staining is distinctly uncommon. Approximately 30% of Ewing sarcomas and PNETs demonstrate diffuse c-kit (CD117) staining.

Several tumors also express CD99 and may confuse the diagnosis. Strong CD99 immunostaining has been observed in ependymoma, glioblastoma, and pancreatic islet cell tumors. These tumors are not typically within the clinical differential diagnosis of Ewing sarcoma or PNET. In contrast, lymphoblastic lymphoma (particularly T-cell), neuroblastoma, alveolar rhabdomyosarcoma, synovial sarcoma, and desmoplastic small round cell tumor may demonstrate variable levels of CD99 staining and are frequently considered in the differential diagnosis of these tumors. Whereas both Ewing sarcoma and lymphoblastic lymphoma may demonstrate strong CD99 staining, Ewing sarcoma and PNET should be negative for lymphocyte markers, particularly leukocyte common antigen and terminal deoxynucleotidyl transferase. Ewing sarcoma and PNET are also more likely to demonstrate strong vimentin staining compared with lymphoblastic lymphoma. The immunohistochemical differentiation of Ewing sarcoma and PNET from neuroblastoma is typically more straight-forward largely because CD99 staining in neuroblastoma, if present, is weak and patchy. Neuroblastomas commonly demonstrate strong staining with NSE, whereas Ewing sarcoma and PNET occasionally will show weak to moderate NSE staining. Poorly differentiated synovial sarcoma and desmoplastic small round cell tumor may both also demonstrate CD99 staining and morphologically be difficult to differentiate from Ewing sarcoma and PNET. In both cases strong membranous staining is more consistent with Ewing sarcoma and PNET. In contrast, weak staining for CD99 and strong staining for cytokeratin and epithelial membrane antigen suggest synovial sarcoma. Desmoplastic small round cell tumors reliably demonstrate nuclear staining for WT1, whereas Ewing sarcoma and PNET typically do not stain with WT1. Nuclear staining for FLI1 may also help differentiate these tumors from other pediatric CD99 small round cell tumors. Most cases of Ewing sarcoma and PNET demonstrate nuclear FLI1 staining, whereas synovial sarcoma, neuroblastoma, and rhabdomyosarcoma only rarely show positive nuclear FLI1 staining. Significantly, however, a high proportion of lymphoblastic lymphomas also exhibit nuclear FLI1 staining. Finally, a number of small round cell tumors of bone harboring novel translocations have been described (see Table 61-1 ). These have recently been classified as Ewinglike sarcomas. As an example, a BCOR/CCNB3- containing tumor has recently been described. This entity has a gene expression profile that is distinct from that of Ewing sarcoma, demonstrates membranous CD99 positivity in only a minority of cases, and can be identified by intense immunohistochemical staining for CCNB3.

The combination of characteristic morphology and immunohistochemistry typically allows an experienced pathologist to render the diagnosis of Ewing sarcoma or PNET. The identification of recurrent chromosomal translocations and their associated oncoproteins in these tumors has allowed for the incorporation of molecular testing into their diagnosis. These methods play a role as confirmatory tests in the setting of typical morphology and immunohistochemistry. In more unusual cases these molecular tests may serve as the key feature on which the diagnosis is based. Although these molecular approaches have been very effective, it is worth recognizing their strengths and weaknesses when interpreting diagnostic studies.

Karyotypic analysis is one approach to confirming the diagnosis of Ewing sarcoma. The presence of one of the characteristic translocations can be considered strong supportive evidence for the diagnosis. It is important to recognize that false-negative results may occur from a number of sources, including overgrowth of nonmalignant elements during culture, complex translocations in which the characteristic Ewing-associated rearrangement is partially masked by the presence of an additional fusion partner, and cryptic rearrangements in which the translocation cannot be identified using typical techniques. FISH can be very helpful as a complementary technique to demonstrate that the EWSR1 locus is “split,” which suggests a translocation event. FISH for the FLI1 and/or ERG loci that demonstrates a split signal from either of these genes or fusion between EWSR1 and FLI1 or ERG signals provides additional strong evidence for involvement of these genes in the rearrangement. A false-negative FISH result may occur when the fusion involves a rarer translocation partner, such as TLS (in lieu of EWS) or ETV1, ETV4, or FEV (in lieu of FLI or ERG). These rare translocations are not typically included in the molecular diagnostic evaluation of these tumors.

A positive EWSR1 “split” FISH assay is not specific for the diagnosis of Ewing sarcoma because of the presence of EWSR1 rearrangements in tumor types other than Ewing sarcoma (see Table 61-1 ). For example, clear cell sarcoma (also called malignant melanoma of the soft parts) usually harbors a t(12;22)(q13;q12) rearrangement. This translocation fuses EWS to the ATF1 protein to form the EWS/ATF1 protein. Similarly, desmoplastic small round cell tumor harbors a t(11;22)(p13;q12) rearrangement. This fuses the EWS protein to the Wilms tumor suppressor WT1 to form the EWS/WT1 protein. EWS or TAF15 can join the NR4A3 orphan nuclear receptor to form EWS/NR4A3 and TAF15/NR4A3 fusion proteins in extraskeletal myxoid chondrosarcoma. EWS is occasionally fused to DDIT3 (also called CHOP) to form EWS/DDIT3 in t(12;22)(q13;q12) positive myxoid liposarcoma. An EWS/ZNF278 fusion was identified in a small round cell sarcoma as a result of a t(1;22)(p36;q12) rearrangement. In some cases there may be diagnostic confusion between some of these tumor types and Ewing sarcoma, and thus the presence of a rearranged EWSR1 locus may not be adequate for diagnostic purposes. In rare instances tumors may show biphenotypic differentiation with both neural and myogenic patterns. At least some of these express EWS rearrangements (EWS/FLI or EWS/ERG fusions) as well as the classic PAX3/FKHR fusion protein associated with alveolar rhabdomyosarcoma.

The use of RT-PCR may provide greater specificity in the diagnosis of Ewing sarcoma. RT-PCR is a useful method for detection of EWS/ETS fusions. As with any PCR-based method, false-positive results may occur from contamination. False-negative results may occur because of degradation of the RNA, alternate fusion partners, or because an RT-PCR primer pair may be designed to detect some EWS/FLI transcripts but not others. False-negative results may also occur when biopsies do not capture viable tissue or primarily include nonmalignant elements. Despite their shortcomings, molecular techniques have become an important addition to the diagnostic approach for Ewing sarcoma.

Local Control

Patients with nonmetastatic Ewing sarcoma require aggressive multimodality therapy to achieve long-term disease control. Local control of the primary tumor plays a key role in modern treatment for these patients. Historically, patients received radiation therapy alone as local control. Surgical local control was less commonly used because therapy was generally viewed as palliative. With improved outcomes thanks to chemotherapy, however, the late effects of radiotherapy on long-term survivors of Ewing sarcoma became apparent. In addition, limb-salvage surgical techniques became more advanced. As a result, surgical local control of resectable tumors has become the standard of care. Radiotherapy for local control of the primary tumor is now generally reserved for unresectable tumors or tumors that have been resected with inadequate margins. This section reviews the principles of radiotherapy and surgical resection in the care of patients with Ewing sarcoma.

Surgical resection is generally recommended for those tumors deemed completely resectable with wide or radical margins. For extremity tumors, surgical resection may take the form of an amputation or a limb-sparing procedure. Rotationplasty is another option for patients with femoral tumors. Technical advances, such as expandable implanted prostheses, have made limb-sparing procedures increasingly appealing. However, the choice of a limb-sparing procedure over other surgical options must not compromise the adequacy of the resection. Tumor infiltration of surrounding neurovascular structures, muscle, or skin should raise concern that a limb-sparing procedure will not result in the desired wide resection. Often neoadjuvant chemotherapy will produce sufficient response to allow for limb salvage when this option did not appear feasible at initial presentation. In a cooperative group analysis of chest wall tumors, resection after neoadjuvant chemotherapy was associated with higher rates of negative margins compared to initial resection. However, data from a single institution suggested that neither the likelihood of an adequate margin nor the overall outcome differs between patients who had surgical resection at initial diagnosis compared with patients who had surgical resection after a period of neoadjuvant chemotherapy. Nevertheless, most available data support the current standard approach of neoadjuvant chemotherapy before planned surgical resection.

Investigators from the CESS group and the Rizzoli Institute evaluated the impact of surgical margins on outcome in Ewing sarcoma in two retrospective case series. These groups both reported the rate of obtaining an adequate surgical margin as approximately 75%, among those patients selected for planned definitive resection. Both groups observed that an adequate margin is more difficult to obtain in axial tumors. For example, the Rizzoli group reported that approximately one quarter of the extremity tumors treated with a limb-salvage procedure had inadequate margins. In contrast, none of the paraspinal or sacral tumors in their large series of surgically treated patients had an adequate margin. The Rizzoli group observed that the 5-year event-free survival rate was higher for patients with an adequate surgical margin, although this finding may reflect improved outcome with smaller, more resectable tumors. In both series a small group of patients with inadequate surgical margins did not receive postoperative radiotherapy. Only 14% to 21% of these patients experienced a local recurrence. However, the risk of local recurrence was diminished by the addition of postoperative radiotherapy, which is the current standard practice.

Radiation therapy alone can provide adequate local control in Ewing sarcoma. In fact, the radiosensitivity of these tumors was one of the early characteristics that distinguished Ewing sarcoma from osteosarcoma. Local control rates with definitive radiation therapy in large series have been reported to range from 53% to 86%. For patients treated with radiation therapy alone, axial tumors (particularly pelvic tumors) have a higher rate of local failure than extremity tumors. One group reported that patients with metastatic disease treated with radiation alone or surgery plus radiation have a higher rate of local failure compared with patients who have nonmetastatic disease.

Several studies have evaluated the appropriate treatment volume, dose, timing, and schedule of radiotherapy in Ewing sarcoma. The IESS-1 study group reported on their experience with radiotherapy. All patients received radiotherapy as the mode of local control in this study. Across the range of doses administered (30 Gy to more than 60 Gy), no differences in local failure rates were observed, although very few patients received less than 40 Gy. Most patients received radiation to the whole bone involved with tumor, but some patients were treated with more limited fields. Patients who received radiotherapy to the primary tumor plus a 5-cm margin had the same local failure rate as patients treated with whole bone radiotherapy (8% for both groups). Patients treated with less than a 5-cm margin had an inferior local control rate.

The Pediatric Oncology Group conducted a study from 1983 and 1988 with the specific aim of determining the appropriate radiation volume for patients with Ewing sarcoma of the bone. Patients with tumors in expendable bones were recommended for surgical resection. Other patients were randomized to receive whole bone irradiation or involved field radiation. Patients treated with whole bone irradiation received 39.6 Gy to the entire bone containing tumor plus a boost to 55.8 Gy to the tumor with a 2-cm margin. Patients treated with involved field radiation received 55.8 Gy to the tumor with a 2-cm margin. Because of enrollment issues, 20 patients were randomized to whole bone radiation and 20 patients to involved field radiation. An additional 54 patients were nonrandomly assigned to involved field radiation. The 5-year event-free survival rate did not differ between patients randomized to whole bone radiation and patients randomized to involved field radiation (37% and 39%, respectively). The local control rate was 53% in both treatment arms. Of note, patients who received radiotherapy according to protocol guidelines for dose and volume had a superior local control rate (80%) compared with patients with major deviations (16% local control rate) or minor deviations (48% local control rate). Deviations in treatment volume seemed to be more critical. These results indicate that involved field radiation is an acceptable mode of local control for these patients but that treatment to the initial tumor volume plus an adequate margin is necessary.

Several retrospective single-institution case series have evaluated the impact of radiation dose on local control in Ewing sarcoma. In one series patients treated with less than 49 Gy had a 5-year local control rate of 37% compared with 89% for patients treated with 49 Gy or more. In this study higher doses seemed particularly important for larger tumors. In another series patients with larger tumors had a uniform local failure rate regardless of radiation dose, whereas patients with smaller tumors treated with more than 40 Gy had a higher local control rate compared with patients who had smaller tumors treated with less than 40 Gy. A third case series reported no difference in outcome between patients treated with radiation doses above and below 54 Gy. The CESS-81 trial randomized extremity tumors to receive 46 Gy or 60 Gy. The local failure rate was similar in these two groups.

Three studies have specifically commented on the use of lower-dose radiotherapy. The first study administered approximately 30 Gy in patients mainly in conjunction with some type of surgical resection, although five patients received radiotherapy only. No local failures were noted in this group. The other two studies administered 30 to 36 Gy as definitive local control to patients with an objective response to neoadjuvant chemotherapy. This strategy resulted in an unacceptably high local failure rate.

The timing of radiotherapy may be important for patients receiving definitive radiotherapy and not for patients receiving radiotherapy after surgery. A retrospective analysis of patients treated with surgery followed by radiation therapy in the CESS-86 and EICESS-92 trials demonstrated that the interval between surgery and radiation did not affect the local control rate or event-free survival rate, even up to intervals beyond 90 days. In contrast, a retrospective analysis of patients with pelvic Ewing sarcoma suggested that initiating definitive radiotherapy earlier may result in a lower local failure rate. A pooled analysis has also suggested that longer periods (15-18 weeks) of neoadjuvant chemotherapy before starting radiation therapy may have a negative impact on the overall survival rate.

Two retrospective case series evaluated hyperfractionated radiotherapy in the management of Ewing sar­coma. Both series concluded that local control rates were not improved by hyperfractionation. One group reported better functional results with the hyperfractionated schedule. The CESS-86 trial randomized patients to conventional or hyperfractionated radiation schedules. Patients on the conventional schedule had chemotherapy held during radiotherapy, while patients on the hyperfractionated schedule continued to receive chemotherapy during radiation. For patients treated with radiotherapy alone, the local control rate was 82% for patients on the conventional schedule and 86% for patients on the hyperfractionated schedule. The radiation schedule did not affect overall or relapse-free survival rates. The radiation schedule did not affect disease control in patients treated with surgery followed by radiation.

On the basis of this experience with radiotherapy for Ewing sarcoma, current practice is for patients with unresectable tumors to receive definitive radiotherapy after 4 to 6 cycles of chemotherapy. Most groups administer 54.4 to 60 Gy as definitive radiotherapy. Patients typically receive treatment as 45 Gy to the pretreatment volume plus a safety margin of at least 2 cm followed by a boost to full-dose radiotherapy to the treatment volume remaining after neoadjuvant chemotherapy. Tumors with soft tissue extension, but not infiltration, into the chest or pelvic cavities often receive treatment to the postchemotherapy volume plus a safety margin. Some centers advise full-dose preoperative radiotherapy to tumors that may become fully resectable with such therapy. Patients with inadequate surgical margins are recommended to receive postoperative radiation. The EURO-EWING group also recommends postoperative radiotherapy for patients with adequate surgical margins and a poor histologic response to chemotherapy, although this practice is not standard in North America. The EURO-EWING group recommends a hyperfractionated schedule of radiotherapy, whereas the standard in North America remains conventional fractionation.

Several groups have compared the three available modes of local control: surgery, definitive radiation, and surgery plus radiation. Several studies have clearly documented that patients who undergo surgical resection have higher local control rates or improved overall outcomes (or both) compared with patients who receive definitive radiotherapy only. * These studies must be interpreted with caution because of the presence of confounding by indication. For example, because small extremity tumors are typically selected for surgery and large axial tumors are typically managed with radiotherapy alone, the outcome between these two groups might be expected to differ on account of reasons other than the chosen mode of local control. Indeed, not all studies have concluded that surgery provides superior disease control compared with radiation. A retrospective single-institution study of 76 patients with nonmetastatic Ewing sarcoma concluded that the overall survival rate and the rate of local control were equivalent between patients who received local control as surgery, radiation, or surgery plus radiation, although the power to detect a difference may have been limited by the small size of this study. A secondary analysis of data from the United Kingdom Children’s Cancer Study Group (UKCCSG) ET-2 trial reached the same conclusion. In addition, the overall rate of disease recurrence did not differ between local control groups. In the SE 91-CNR Italian cooperative group trial, the 3-year event-free survival rates did not differ between patients who received surgery alone and those who received radiotherapy alone.

* References .

Systemic Treatment with Chemotherapy

The use of chemotherapy for patients with Ewing sarcoma has revolutionized the care of these patients. Before the 1970s, standard therapy for this disease consisted solely of local control directed at the primary tumor. Most patients treated in this manner ultimately died from local or systemic recurrent disease. In one series patients treated with local control alone had a median survival rate of 11 months. In the 1960s and 1970s, evidence emerged that systemic chemotherapy could prevent recurrent disease in a substantial number of patients with Ewing sarcoma. This section reviews the development of modern chemotherapy regimens for this disease. Table 61-3 lists major chemotherapy regimens evaluated for Ewing sarcoma along with abbreviations for these regimens. Table 61-4 summarizes the major cooperative group trials that have contributed to current treatment approaches to this disease.

Management of Tumors Arising at Special Sites

Summary of Outcomes for Patients with Nonmetastatic Ewing Sarcoma

Patients with Ewing sarcoma have shown great improvements in outcome over the past 4 decades. For patients with localized tumors, expected survival rates have increased to greater than 70% with modern multimodality therapy. Figure 61-8 demonstrates these improvements graphically, using Surveillance, Epidemiology, and End Results (SEER) data from 1975 to 2000. A similar pattern of steady improvement over time has also been reported using data from a large European registry.

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

The Neonatal Erythrocyte and Its Disorders Diagnostic Approach to the Anemic Patient Approach to the Child with a Suspected Bleeding Disorder Disorders of the Red Cell Membrane Pediatric Myeloid Leukemia, Myelodysplasia, and Myeloproliferative Disease Nonrhabdomyosarcoma Soft Tissue Sarcomas and Other Soft Tissue Tumors

Stay updated, free articles. Join our Telegram channel

Join