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Molecular Biology of the Ewing’s Sarcoma/Primitive Neuroectodermal Tumor Family

From the Department of Pathology, Clínica Universitaria de Navarra, Pamplona, Spain, and Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY.

Address reprint requests to William L. Gerald, MD, PhD, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021; email geraldw{at}mskcc.org

ABSTRACT

ABSTRACT: Ewing’s sarcoma (ES) and primitive neuroectodermal tumor (PNET) are members of a tumor family consistently associated with chromosomal translocation and functional fusion of the EWS gene to any of several structurally related transcription factor genes. Similar gene fusion events occur in other mesenchymal and hematopoietic tumors and are tumor-specific. The resulting novel transcription factor–like chimeric proteins are believed to contribute to tumor biology by aberrant regulation of gene expression altering critical controls of cell proliferation and differentiation. These tumor-specific molecular rearrangements are useful for primary diagnosis, may provide prognostic information, and present potential therapeutic targets. The recent advances in our understanding of the molecular biology of ES and PNET represent a paradigm for the application of the basic biology of neoplasia to clinical management of patients.

EWING’S SARCOMA (ES) and primitive neuroectodermal tumor (PNET) were described in the early part of this century as distinct clinicopathologic entities. Arthur Purdy Stout¹ described a tumor of the ulnar nerve with the gross features of a sarcoma but composed of small round cells focally arranged as rosettes (subsequently designated peripheral PNET). James Ewing² described an undifferentiated tumor in the diaphysis of long bones that was radiosensitive (ES). In ensuing years, the overlapping characteristics of these two categories of small round-cell tumor have become well known, supporting the concept of a single tumor category with variable phenotypic expression and leading to the acceptance of a unified diagnostic grouping. The ES/PNET family also includes several other related clinicopathologic neoplastic entities, such as malignant small-cell tumor of the thoracopulmonary region (Askin’s tumor), paravertebral small-cell tumor, atypical ES, PNET of bone, and extraosseous ES.^3-7 These tumors are poorly differentiated, small, round-cell tumors and often pose difficult diagnostic problems when examined by light microscopy alone. For that reason, they have become a model system for nonmorphologic approaches to diagnosis and subclassification, such as immunophenotypic and genetic analysis. From this multimodal investigation have come improved diagnostic criteria and a better understanding of their basic biology. In addition to sharing some clinical and morphologic features, these malignant neoplasms have common genetic, in vitro, and immunophenotypic characteristics that further validate the concept of a single, genetically defined tumor category.⁸ Advances in our understanding of the basic biology of the ES/PNET family have had a direct impact on clinical care and places this tumor family at the forefront of intensive efforts to understand the relationship between genotype, tumor biology, and clinical phenotype.

CLINICAL AND PATHOLOGIC FEATURES

ES and PNET affect primarily white and Hispanic young people and is extremely rare in individuals of African or Asian origin. The reason for this striking ethnic distribution is not known, although interethnic differences exist for certain alleles of one of the genes consistently disrupted in ES and PNET.⁹ Tumors can develop in almost any bone and soft tissue and often present with pain and swelling. Approximately 25% of patients have detectable metastatic disease to lung, bone, and bone marrow at diagnosis, but nearly all patients have micrometastases, as evidenced by a 10% cure rate with local therapy alone. The standard of care is systemic therapy combined with surgery or radiotherapy for local control. Despite advances in therapy, 5-year survival rates are only approximately 50%. The most important negative prognostic factor is tumor dissemination at the time of diagnosis.¹⁰

Typical histologic features include sheets of monomorphic round cells with small hyperchromatic nuclei, inconspicuous nuclei, and scant cytoplasm. There is usually extensive necrosis with peritheliomatous preservation of viable tumor and a lack of intercellular material. Variants include larger cells, greater pleomorphism, more condensed chromatin, and a lobular or organoid growth pattern. Tumors can exhibit a variable degree of neural differentiation, but this is usually subtle and often only detected immunophenotypically. The histogenesis of ES and PNET is unknown; however, a neuroectodermal origin has been proposed based on variable expression of neuronal immunohistochemical markers, ultrastructural features, and the ability of ES/PNET cell lines to differentiate along a neural pathway in vitro.¹¹ Nevertheless, other histogenetic possibilities cannot be excluded because ES and PNET can also exhibit some epithelial and mesenchymal characteristics and can arise in organs not directly related to the neural crest (eg, the kidney).

No routinely used histochemical or immunohistochemical stains can positively distinguish ES and PNET from other undifferentiated tumors of childhood; however, in the vast majority of cases, ES and PNET have been shown to express at extremely high levels an antigen determined by the MIC2 gene.^12,13 The product of MIC2 is a glycoprotein (also designated CD99 or p30/32^MIC2) with a molecular mass of approximately 30,000 daltons located on the cell surface and believed to be involved in cell adhesion. Although immunohistochemical detection of membrane-localized MIC2 expression is a sensitive diagnostic marker for the ES/PNET family of tumors, it lacks specificity in that many other tumors, and for that matter, many normal tissues, are also immunoreactive with anti-MIC2 antibodies in some cases (Table 1).¹⁴ The diagnosis of ES and PNET has been largely a process of exclusion. In recent years, detection and investigation of specific genetic alterations have established exquisitely sensitive and specific markers for ES and PNET that have rapidly become the standard for confirming the diagnosis.

Consistent Chromosomal Translocations in ES and PNET
An important advance in the study of mesenchymal and hematopoietic tumors has been the identification of consistent chromosomal translocations associated with unique tumor types (Table 2).¹⁵ These translocations interrupt specific genes and recombine them to create novel fusion genes. The fusion genes are expressed and encode proteins that combine functional domains usually found in separate molecules. Because the fusion gene and its products are tumor-specific and present in virtually all cases of an individual tumor category, their characterization not only yields profound insights into tumor biology but also holds great promise for new diagnostic and therapeutic approaches.

View larger version (21K): [in this window] [in a new window]   Fig 1. (A) Schematic diagram of t(11;22)(q24;q12) consistently associated with ES/PNET. (B) Schematic diagram of EWS-FLI1 gene fusion resulting from t(11;22). Individual gene exons are depicted as numbered boxes, and the line represents intervening and flanking DNA. The regions where chromosomal breakpoints occur are bracketed.

Translocation Partners of EWS in ES and PNET
The FLI1 gene on 11q24 was the first EWS translocation partner identified in the ES/PNET family of tumors. It is a member of a large family of DNA-binding transcription factors that are implicated in the control of cellular proliferation, development, and tumorigenesis.^32-34 The family members are defined by the presence of a highly conserved 85-amino acid domain termed the erythroblastosis virus–transforming sequence (ETS) domain. This domain mediates specific binding to purine-rich DNA sequences characterized by a GGA(A/T) core element.³⁵ In ES/PNET-associated translocations, the ETS domain can be contributed from any of several family members. In 85% of cases, it is derived from FLI1 [t(11;22)(q24;q12)], and in 10% of cases, it is from ERG (ets-related gene; t(21;22)(q22;12)).^36,37 In rare instances, EWS is fused to the ETS domains of ETV-1 (ets translocation variant 1; t(7;22)(q22;12)), E1AF (E1A factor; t(17;22)(q12;q12)), or FEV (fifth Ewing variant; t(2;22)(q33;q12)).^38-40 The functional consequences of these less frequent variant translocations are thought to be analogous to the EWS-FLI1 rearrangement, but subtle differences are likely to exist.

ETS domain–containing proteins act as either transcriptional activators or repressors. These functions are modulated by a combination of specific DNA-protein and protein-protein interactions. ETS family members have been shown to cooperate with other nuclear proteins and often function as part of a larger protein complex that helps to establish promoter specificity, modulates transcriptional regulation, and allows linkage to signal transduction pathways.^32-34 Many ETS domain proteins are targets of signal transduction pathways and are activated in response to a wide array of extracellular stimuli. For example, activation of RAS leads to mitogen-activated protein kinase–mediated phosphorylation of some ETS proteins.⁴¹ Because RAS is implicated in cell-cycle regulation and tumor angiogenesis and is permanently activated in a high percentage of human cancers, it may be that RAS-responsive ETS proteins play a general role in oncogenesis.

ETS domain proteins are implicated in tumors other than ES and PNET.³⁴ Chromosomal translocation and fusion of the ETS domain of ERG to TLS/FUS is present in myelocytic leukemia, and fusion of the carboxy- or amino-terminal region of TEL to a variety of translocation partners occurs in some myelocytic and lymphoblastic leukemias. The TEL translocation partners include AML1, ABL, PDGFR-beta, JAK2, and MN1. The amino-terminal region of TEL does not contain the ETS DNA-binding domain but has a motif that mediates protein-protein interactions. The oncogenic role of TEL amino-terminal region–containing fusion proteins seems to be mediated by homotypic oligomerization and activation of gene products involved in growth-related pathways. These two different functional domains present in ETS proteins can contribute to tumorigenesis through unique mechanisms.

Translocation Partners of EWS in Other Tumor Types
Although translocations involving the EWS gene and ETS family genes only occur consistently in ES and PNET, fusions of EWS to different transcription factor genes have been observed in other tumors (Table 1). EWS is fused to ATF1 in malignant melanoma of soft parts, WT1 in intra-abdominal desmoplastic small round-cell tumor, CHOP in myxoid liposarcoma, and CHN in myxoid chrondrosarcoma.^42-46 In addition, an EWS-like gene, TLS/FUS, is involved in tumor-associated gene fusions in myxoid liposarcoma (TLS/FUS-CHOP) and acute myeloid leukemia (TLS/FUS-ERG).^21,22,47,48 Chimeric proteins in this group of tumors have a structure analogous to that of EWS-FLI1 described above and presumably a similar function. All result in the fusion of the amino-terminal portions of EWS or TLS/FUS to DNA-binding domains of transcription factors, further suggesting that disruption of transcriptional control contributes to their transforming potential. It is important to realize that in each case, with few exceptions, a distinct clinicopathologic tumor entity has been found to be consistently associated with a translocation involving a unique class of transcription factor. This tumor specificity is most likely related to cell-specific influences on DNA recombination, chimeric gene expression, and intrinsic proteins that are necessary to complement chimeric protein action.

Other Genetic Alterations in the ES/PNET Family
The consistent EWS-ETS gene family rearrangements present in the ES/PNET family indicate that they are a primary and probably rate-limiting step in tumorigenesis; however, other genetic alterations occur and may contribute to tumor biology. By conventional and molecular cytogenetics, frequent gains of chromosomes 8 and 12 and losses of 1p and an unbalanced der16t(1;16) have been identified.^49,50 These regions may contain genes that play a role in ES/PNET progression. In addition, changes in several known tumor-associated genes occur in some cases. Homozygous deletion of CDKN2A (P16, INK4) has recently been described in 18% to 30% of primary cases, and mutations of p53 are detected in 5% to 20%.^51-53 Mutations affecting these two genes are more frequent in ES/PNET cell lines, indicating a selection bias for growth in culture.

CHIMERIC MOLECULE FUNCTION

Regulation of Transcription
The ES/PNET-associated translocations result in chimeric products containing the amino-terminal domain of EWS fused to the nucleic acid–binding domain of the transcription factor translocation partner (Fig 2). This structure suggests that the chimeric protein is directed to the promoter region of specific genes recognized by the translocated DNA-binding domain and contributes to tumor development by altering expression of these genes. In support of this, some EWS fusion products have been shown experimentally to function as transcriptional activators.^54-57 The EWS-FLI1 protein localizes to the nucleus and can bind DNA in a site-specific manner via the ETS domain. The DNA-binding site specificity and affinity of EWS-FLI1 are similar to those of native FLI1, which suggests that the chimeric protein might interact with the same genes normally regulated by FLI1. However, the targets of action are not solely dependent on the ETS domain and may be affected by protein-protein interactions that are unique to the chimeric molecule and result in a different spectrum of targets.⁵⁸ EWS-FLI1 has been shown to be an efficient transcriptional activator when cotransfected with model reporter gene constructs containing ETS-binding sites in their promoters. This transcriptional activation function is dependent on the presence of both the EWS and FLI1 portion of the chimeric molecule. The data suggest two possible mechanisms by which chimeric transcription factors may contribute to tumor biology: (1) EWS-FLI1 may affect transcription of some of the same genes normally regulated by native FLI1, but because the chimeric molecule is expressed in a different temporal or quantitative manner, the downstream targets are deregulated, affecting the growth of cells; and (2) EWS-FLI1 may affect target genes different from those regulated by native FLI1, which would explain why overexpression of FLI1 does not have the same transforming properties as overexpression of EWS-FLI1.⁵⁹ These two mechanisms are not mutually exclusive.

View larger version (13K): [in this window] [in a new window]   Fig 2. Schematic diagram of the chimeric EWS-FLI1 protein product (NTD, amino-terminal domain; BD, binding domain).

Cell Transformation
The consistent presence of these chimeric molecules in the ES/PNET family suggests a critical role in tumor biology. In experiments to evaluate the ability of fusion proteins to cause neoplastic transformation, the chimeric protein has been shown to affect the growth characteristics of some cell lines. Transfection of EWS-FLI1 or EWS-ERG can transform mouse NIH3T3 fibroblasts so that they acquire tumor-like properties, such as growth in soft agar and immunodeficient mice.^54,59 Both the EWS and FLI1 domains are required for transforming activity. Transfection of wild-type FLI1 does not result in cell transformation, which suggests that EWS-FLI1 is functionally distinct. Not all cell types are efficiently transformed by EWS-FLI1, which suggests that chimeric molecule function is dependent on a permissive cellular background. This may indicate that other cellular proteins modulate the function of EWS-FLI1. In this regard, it has been shown that EWS-FLI1 is able to cooperate with other transcription factors to activate a model reporter gene and that expression of insulin-like growth factor 1 receptor is required for NIH3T3 transformation.^68,69 Cell context may, therefore, have an important bearing on the role of tumor-specific chromosomal translocations, as described above.

The fusion protein has also been shown to be important for maintaining the growth characteristics of ES/PNET cell lines. EWS-FLI1 RNA antisense molecules transfected into ES/PNET cells decrease expression of EWS-FLI1 and result in growth inhibition in culture and tumor xenografts.^70,71 In addition, truncated ETS domain–binding molecules can act as competitive inhibitors and have a dominant negative effect on cell growth.⁷² This implies that a certain level of expression of the chimeric molecule and its downstream targets is necessary for the growth-related affects of EWS-FLI1 and suggests a promising field of investigation for novel therapeutic approaches.

EWS-FLI1 may also exert tumorigenic effects via deregulation of programmed cell death. Cancer is believed to result in part from an imbalance in cell growth and cell death. Expression of EWS-FLI1 or EWS-ERG in NIH3T3 cells inhibits apoptosis that would normally occur with serum deprivation or calcium ionophore treatment.⁷³ In addition, antisense inhibition of EWS-FLI1 results in increased susceptibility to chemotherapy-induced apoptosis in ES/PNET cell lines. These data suggest that chimeric molecules may affect mechanisms of programmed cell death and thereby play a role not only in tumor development but also in response to therapy.

MOLECULAR DIAGNOSTICS

Routine techniques in surgical pathology allow the diagnosis of most cases of ES and PNET, but genetic analysis is very helpful when morphology is not conclusive or tumors present in unusual clinical settings.^74-76 Standard cytogenetic studies can reveal a wide variety of chromosomal alterations, but technical constraints and variant translocations may make interpretation difficult, and results are often obtained too late to influence therapy. Molecular assays for specific chromosomal changes, such as those found in ES and PNET, are attractive for several reasons. Two clinically important features are the small sample size requirement and the ability to provide results rapidly. Such studies are also considered more sensitive than traditional cytogenetics, with a higher technical success rate for detecting specific gene fusions and the potential to detect cryptic translocations. A particularly appealing advantage for diagnostic molecular studies is that, despite distortions of histologic patterns or the absence of histologic or immunohistochemical evidence of differentiation, the basic molecular genetic alterations are present in tumor cells and molecular analysis may permit greater confidence in a specific diagnosis.

Many molecular techniques have proven useful for detection of the EWS translocations associated with ES and PNET, including fluorescent in situ hybridization, Southern blotting, DNA-based polymerase chain reaction (PCR), and RNA-based PCR (also referred to as reverse transcriptase [RT]-PCR).⁷⁷ The translocations associated with ES and PNET can be readily identified using RT-PCR by the presence of fusion transcripts in tumor cells, and this technique has become a mainstay in molecular diagnosis. RT-PCR is extremely specific and sensitive and allows detection of very low levels of tumor cells even when they present among large numbers of normal cells, such as circulating tumor cells in peripheral blood or bone marrow. This provides an extremely robust method for molecular staging and monitoring treatment response. Stage of disease at diagnosis as determined by standard imaging modalities constitutes the most powerful predictor of prognosis for patients with ES or PNET. Some patients considered to have localized disease, however, may have unfavorable outcome due to minimal metastatic disease not detected by traditional methods. Several groups have demonstrated that minimal disease can be detected in peripheral blood and bone marrow using PCR-based methods and may contribute to patient management. Nevertheless, a number of technical, biologic, and clinical questions remain.^78-82

Molecular Markers of Prognosis
In ES and PNET, several factors have been considered to be of prognostic importance (stage, primary tumor site, size, age, and response to therapy).¹⁰ Recent studies have evaluated the contribution of molecular heterogeneity in the ES/PNET family to prognosis.^50,83-85 Gene fusions in ES and PNET show molecular variability, with at least 18 different structural possibilities. There are two sources of variability: the fusion partner (FLI1, ERG, ETV1, E1A, or FEV) and the breakpoint location within the genes. A better outcome for patients with localized tumors expressing the most common chimeric transcript (EWS exon 7 fused to FLI1 exon 6) compared with other fusion types has been reported. This raises the possibility that heterogeneity in chimeric transcript structure may reliably define clinically distinct risk groups. The biologic basis for the prognostic difference between EWS fusion types is unknown, but a recent report has shown functional differences among the various fusion genes.⁸⁶ Direct comparison between treatment response and EWS fusion type may reveal a role of certain chimeras in therapy resistance. These reports should stimulate inclusion of EWS fusion-type determination into prospective studies.

Studies evaluating the prognostic significance of other cytogenetic and molecular alterations in ES/PNET are limited. However, del1p, homozygous deletion of CDKN2A, and TP53 mutation have all been associated with a worse prognosis.⁵⁰ The association of genetic variants of ES and PNET with clinically significant features is an area that deserves further study.

Therapeutic Potential of Molecular Targets
Identification of molecular characteristics of the ES/PNET family has contributed to clinical care by providing a greater degree of confidence for an often difficult diagnostic problem. An equally exciting area of current investigation is the exploitation of these tumor-specific biologic properties as targets for novel therapeutic approaches. Current efforts have focused on immunotherapy directed at tumor cell–specific epitopes derived from chimeric molecules, but approaches to directly inhibit chimeric proteins and their downstream targets also seem feasible.⁸⁷ Further understanding of the basic biology of these tumor-specific molecules has the potential to lead to novel therapies.

In summary, identification and characterization of the EWS-FLI1 fusion in the ES/PNET family of tumors further support the oncogenic role of transcription-modulating chimeric proteins resulting from chromosomal translocations. The elucidation of specific pathogenetic contributions of fusion genes will give insight into the development and maintenance of the malignant phenotype for this family of tumors. Of practical importance, further investigation of the structural and functional attributes of the EWS-FLI1 gene fusion has begun to provide the necessary basic information to directly benefit individuals with ES and PNET.

ACKNOWLEDGMENTS

Supported in part by grant no. RO1 CA68273 (to W.L.G.) from the National Institutes of Health and FIS 99/0646 from the Health Care System of Spain (to E.dA.).

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Submitted August 2, 1999; accepted September 28, 1999.

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