Ewing's Family Research Articles

Morphologic and Immunophenotypic Diversity in Ewing Family Tumors

More than 85% of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), or "Ewing family of tumors" (EFTs), have the translocation, t (11;22) (q24;q12), with others having variant translocations. Identification of these by cytogenetic and/or molecular genetic techniques is specific for EFT and is increasingly recognized as the "gold standard" for diagnosis. However, these techniques are not universally available. We therefore studied a large group of genetically confirmed EFTs to more completely understand the morphologic and immunophenotypic spectrum of this rare sarcoma. Sixty-six cytogenetically, FISH or RT-PCR proven-EFTs were retrieved. In 56 cases, immunohistochemistry (IHC) was performed for pan-cytokeratins (PanCK), high molecular weight cytokeratins (HMWCK), desmin (DES), CD99, CD117, and FLI1 protein using heat-induced epitope retrieval and the Dako Envision system. The cases arose chiefly in children and young adults (median 18 years; range, 3-65 years) of both sexes (male, 32; female, 31; unknown, 3) in a variety of bone (N = 39) and soft tissue (N = 27) sites. Histologically, 46 cases (73%) showed only typical features of ES, 9 cases (16%) showed features of PNET, 3 cases (5%) showed "adamantinoma-like" features, 3 cases (5%) corresponded to "atypical Ewing sarcoma," 3 cases (5%) showed principally intersecting fascicles of spindled cells, and 2 cases had abundant hyalinized matrix. IHC results were as follows: PanCK (18 of 56, 32%), HMWCK (3 of 55, 5%), DES (1 of 56, 2%), CD99 (52 of 52, 100%), CD117 (13 of 54, 24%), and FLI1 (44 of 47, 94%). HMWCK was expressed only in "adamantinoma-like" EFTs, none of which expressed DES. In conclusion, most, but not all, EFTs can be accurately diagnosed using time-honored morphologic criteria and ancillary immunohistochemistry. However, genetic confirmation remains essential for the diagnosis of unusual morphologic variants of EFT, including "adamantinoma-like," spindled, sclerosing, and clear cell/anaplastic variants. Therefore, to exclude or confirm the diagnosis of Ewing's sarcoma in round cell sarcomas having a variety of patterns but not specifically conforming to a tumor of known lineage (eg, rhabdomyosarcoma), cytogenetics, and/or molecular analysis is required.

Structure and function of ETAA16: a novel cell surface antigen in Ewing's tumours.

Immunoscreening of an Ewing's family of tumour (EFT)-derived cDNA library using formerly described EFT-specific antibodies led to the isolation of a 3.5 kb cDNA, named Ewing's tumour-associated antigen 16 (ETAA16). The ETAA16 cDNA shows no homology to any functionally characterised human gene. Only a bovine cDNA expressed in bovine testis and hepatocytes is functionally characterised as it encodes for a junction plaque associated protein and showed a homology of 69.9% at amino acid level to ETAA16. The human cDNA encodes for a 926 amino acid tumour antigen with a calculated molecular weight of 103 kDa. The epitope of the ETAA16-specific antibody, Ak16, covers the central region of the protein which is part of an extra cellular domain. The human ETAA16 gene locus has been assigned to chromosome 2p13-15 by FISH analyses and is confirmed by the human genome sequencing project. As demonstrated by flow cytometry, the cell surface expression of ETAA16 antigen is restricted to ET cell lines and not expressed on other small blue round cell tumours or other kind of tumour. RT-PCR analysis revealed a high expression of ETAA16 in brain, liver and kidney while lung and heart were negative. Immunohistochemistry showed an intracellular expression of ETAA16 in different kind of non-Ewing tumour tissues. These results suggest that ETAA16 may function as a tumour-specific cell surface antigen in EFTs.

Low Levels of Her2/neu Expressed by Ewing's Family Tumor Cell Lines Can Redirect Cytokine-Induced Killer Cells

To identify novel treatments for pediatric solid tumors and/or for malignancies with low-level Her2/neu expression. Using fluorescence-activated cell sorting and immunohistochemistry, Her2/neu expression was determined on cell lines derived vfrom Ewing's family tumors (EFT) and neuroblastoma. Sensitivity to trastuzumab treatment was investigated using an in vitro proliferation assay. Cytotoxicity against EFT cell lines was done with either freshly isolated or ex vivo activated and expanded T cells (cytokine-induced killer cells, CIK cells), with or without addition of a CD3xHer2/neu bispecific antibody. The effects of either trastuzumab, CIK cells alone, or CD3xHer2/neu bispecific antibody redirected CIK cells was determined using a SCID/hu model of EFTs and serial, noninvasive bioluminescent imaging. EFT cell lines express 5- to 10-fold lower levels of her2/neu than either breast (BT-474) or ovarian (SK-OV-3) cell lines. Treatment of EFT cell lines with trastuzumab did not induce growth inhibition either in vitro or in vivo. In contrast, Her2/neu could be used to redirect CIK cell to mediate cytotoxicity against EFTs both in vitro and in vivo (using two different treatment schemas). CD3xHer2/neu bispecific antibody and CIK cells may be a suitable approach to treat malignancies with low-level Her2/neu expression not responsive to trastuzumab.

Type 1 (11; 22)(q24: q12) translocation is common in Ewing’s sarcoma/ peripheral neuroectodermal tumour in south Indian patients

The Ewing's sarcoma family can present diagnostic difficulties. In the past the basis of diagnosis has been a exclusion. Identification of a specific translocation especially t(11;22) (EWS-FLI 1 fusion gene), which is seen in nearly 85 percent of Ewing's sarcoma cases can help in precise diagnosis. We have carried out a study on twenty patient samples diagnosed to have Ewing's sarcoma/peripheral neuroectodermal tumour (PNET)/small round cell malignant tumour. The study involved RT-PCR analysis for the fusion transcript, followed by sequencing to identify the specific type of fusion. Ninety percent (18/20) of the samples tested were found to be t(11;22) translocations involving EWS-FLI 1 genes. Sixty-one percent (11/18) were found to be type 1 fusion and seven were type 2 (39 percentage). This is the first study in India with quantitative information about the types of EWS-FLI 1 translocations present in Ewing's family of tumours in south Indian patients.

EWS-FLI1 Fusion Protein Up-regulates Critical Genes in Neural Crest Development and Is Responsible for the Observed Phenotype of Ewing's Family of Tumors

Tumor-specific translocations are common in tumors of mesenchymal origin. Whether the translocation determines the phenotype, or vice versa, is debatable. Ewing's family tumors (EFT) are consistently associated with an EWS-FLI1 translocation and a primitive neural phenotype. Histogenesis and classification are therefore uncertain. To test whether EWS-FLI1 fusion gene expression is responsible for the primitive neuroectodermal phenotype of EFT, we established a tetracycline-inducible EWS-FLI1 expression system in a rhabdomyosarcoma cell line RD. Cell morphology changed after EWS-FLI1 expression, resembling cultured EFT cells. Xenografts showed typical EFT features, distinct from tumors formed by parental RD. Neuron-specific microtubule gene MAPT, parasympathetic marker cholecystokinin, and epithelial marker keratin 18 were up-regulated. Conversely, myogenesis was diminished. Comparison of the up-regulated genes in RD-EF with the Ewing's signature genes identified important EWS-FLI1 downstream genes, many involved in neural crest differentiation. These results were validated by real-time reverse transcription-PCR analysis and RNA interference technology using small interfering RNA against EWS-FLI1 breakpoint. The present study shows that the neural phenotype of Ewing's tumors is attributable to the EWS-FLI1 expression and the resultant phenotype resembles developing neural crest. Such tumors have a limited neural phenotype regardless of tissue of origin. These findings challenge traditional views of histogenesis and tumor origin.

70. Systemic Administration of siRNA Against EWS-FLI1 Using a Targeted, Non-Viral Formulation Inhibits Growth in a Disseminated Murine Model of Ewing's Sarcoma

Despite aggressive therapy, 40% of patients with localized Ewing's family of tumors (EFT) and 80% of patients with detectable metastasis die due to tumor progression. More specific and effective treatments are needed, especially for high-risk cases. EWS-FLI1, the chimeric gene product of a chromosome translocation found in more than 90% of EFT, is crucial for maintaining EFT tumor growth. Endogenous expression of siRNA targeting the breakpoint of the EWS-FLI1 fusion gene (siEFBP) from a pol III promoter has previously been shown to reduce EWS-FLI1 transcript levels up to 80% and resulted in growth inhibition of the cultured EFT cell line TC135. Since EWS-FLI1 expression is tumor-specific, it serves as an excellent candidate for targeted therapy of EFT, especially metastatic disease.

Aberrant laminin beta-3 isoforms downstream of EWS-ETS fusion genes in ewing family tumors

Ewing family tumors (EFTs) are associated with a chromosomal translocation resulting in a fusion of the amino-terminus of EWS with the DNA-binding domain of an ETS transcription factor. Although previous reports suggested that these chimeric proteins would act as aberrant transcription factors, their downstream targets have not been fully elucidated. To identify downstream targets of these EWS-ETS fusion proteins, we introduced EWS-ETS fusion constructs into a human fibrosarcoma cell line, HT-1080, by retroviral transduction. Here we report that the LAMB3 gene encoding the beta-3 chain of basement membrane protein laminin-5 is induced to a significantly higher level in cells expressing EWS-ETSs than in cells expressing normal ETSs. Additionally through use of an antisense oligonucleotide for EWS-ERG in the W-ES EFT cell line, laminin beta-3 protein was reduced coordinately with EWS-ERG fusion protein expression. Furthermore, we found small mRNAs were preferentially transcribed from the LAMB3 gene in EFT cell lines. Molecular cloning of the entire coding region shows that the alternative transcripts from different promoter(s) located within the intron 14, which encode small proteins, likely are major products of the LAMB3 gene in EFT cells. We show that the small isoforms conferred increased anchorage-independent proliferation to NIH3T3 cells. Together with previous studies showing that laminin-5 is involved in the invasive and malignant phenotype of several tumor types, our data suggest that the oncogenic effect of EWS-ETS may be mediated in part by up-regulation of LAMB3 expression.

Current treatment for Ewing’s sarcoma

Ewing’s sarcoma is the second most common primary bone tumor seen in children and adolescents, and was described by James Ewing in 1921 as a diffuse endothelioma of bone. It is one of the differential diagnoses of pediatric small round blue cell tumors. This is not a single condition, but a group of morphologically and clinically closely related disorders with similar molecular biology – expression of tumor-specific chimeric oncoproteins through balanced chromosomal translocations involving the EWS gene – often referred to as the Ewing family of tumors. This includes Ewing’s sarcoma of bone, extra-osseous Ewing’s sarcoma, Askin tumor and peripheral neuroectodermal tumor. These are aggressive neoplasms with almost 25% of patients having clinically evident metastases at presentation. Ewing’s sarcoma has therefore been considered as a systemic disease necessitating local as well as systemic treatment. An aggressive multidisciplinary approach has resulted in significant improvement in prognosis for patients with these tumors. Despite aggressive treatment, 20–40% of patients with localized disease and almost 80% of patients with metastatic disease at presentation succumb to the illness. Advances in understanding the molecular biology of these tumors will hopefully result in the development of novel treatment approaches. The aim of this article is to review the existing treatment methods and to highlight the more recent approaches to the treatment of this condition.

High-Dose Therapy and Stem-Cell Rescue for Ewing's Family of Tumors in Second Remission

The use of adjuvant multiagent chemotherapy and improved local control with surgery, with or without radiotherapy, has significantly improved the outcome for patients with Ewing’s sarcoma family of tumors (ESFT). The prognosis for patients who present with detectable metastatic disease or who recur remains poor. 1-3 Some of the patients in both of these poor prognosis groups are cured by conventional therapy. What is it that makes these patients different? The answer to that question remains elusive, and attempts to identify factors predictive of survival have been challenging. A recent analysis from St Jude Children’s Research Hospital correlated improved survival with a prolonged initial relapse-free interval (RFI), which was defined as more than 24 months. 4 In this issue, Barker et al 5 report on intensive chemotherapy followed by autologous stem-cell reconstitution (high-dose therapy [HDT]) as consolidation therapy for patients with ESFT in second remission. They conclude that HDT is a potentially efficacious therapeutic modality, at least in a subset of patients. Determining the setting in which HDT is most effective remains controversial because most HDT strategies reserve transplantation for patients who achieve either a partial or complete response. The authors attempt to adjust for this bias by performing a multivariate analysis that controls for RFI and response to therapy. They report that patients with a prolonged RFI and responsive disease and those patients receiving HDT have an improved progression-free and overall survival. These results are provocative, but they still raise the following question: what is the role of HDT in patients with ESFT? Because patients with ESFT have tumors responsive to alkylator-based therapy and these agents are presumed to have a steep dose-response curve, investigators have been intrigued by the possible role for HDT in these tumors. However, unequivocal demonstration of a steep doseresponse curve for alkylating agents in Ewing’s sarcoma is lacking. Review of the available data, however, reveals that HDT followed by autologous stem-cell reconstitution has only been successful for pediatric patients with metastatic neuroblastoma and relapsed lymphoma. 6,7 Although HDT has been used for patients with high-risk ESFT in first remission and for patients at relapse, critical review of the literature is complicated by small sample sizes in predominantly retrospective reviews, the use of different inclusion criteria, and the use of different preparative regimens. 8-12 The Children’s Cancer Group prospectively evaluated the use of HDT for patients with ESFT metastatic to bone and bone marrow. In this subset of patients, using an intent-totreat analysis, the authors did not find a benefit to the use of this modality, with an event-free survival rate of 17%. 3 Further evaluation of the use of HDT at Memorial SloanKettering Cancer Center also concluded that HDT did not improve the outcome for patients with metastatic ESFT. 13 Definite conclusions regarding the role of HDT in ESFT will only be possible from the results of a randomized controlled trial; the EURO-Ewing’s 99 study, which is currently ongoing, is evaluating the role of HDT for patients with ESFT and high-risk disease or lung metastases. This group of patients in high-risk first remission is different from the group of patients reported by Barker et al 5 who were in second remission, but demonstration of efficacy in a prospective randomized trial would certainly lend support to the use of HDT in that group as well.

Identification of CBFA1-regulated genes on SaOs-2 cells

Current knowledge about mechanisms controlling osteoblast-specific gene expression has led to the identification of Cbfa1 as a key regulator of osteoblast differentiation. Several essential questions about this transcription factor remain to be addressed, e.g., the nature of stimuli that may modulate its own expression, as well as the genetic repercussions following alterations in Cbfa1 levels. To identify such Cbfa1-responsive genes, the SaOs-2 cell line was stably transfected with a dominant negative mutant of Cbfa1 (DeltaCbfa1). Comparison of gene expression patterns by differential display on selected SaOs-2 clones allowed the identification of four new genes that may be under the control of Cbfa1. Three of them, SelM, elF-4AI, and RPS24, seemed to be linked to a global change in cellular metabolism and cell growth. The fourth, the CD99/MIC2 gene, was strongly overexpressed (around tenfold) in cells presenting high levels of Deltacbfa1. This observation adds evidence to show that this marker of Ewing family tumors is linked to the osteoblast lineage. The exact function of CD99 remains largely undefined, and this is the first time that its regulation by an essential transcription factor involved in osteoblast differentiation has been observed.

Transactivation of cyclin E gene by EWS-Fli1 and antitumor effects of cyclin dependent kinase inhibitor on Ewing's family tumor cells

Chromosomal translocation t(11; 22)(q24; q12) is detected in approximately 90% of Ewing's family tumors (EFTs) including Ewing's sarcoma and primitive neuroectodermal tumor. This results in the formation of the EWS-Fli1 fusion gene, which produces EWS-Fli1 fusion protein. This chimerical gene product acts as an aberrant transcriptional activator, which may be responsible for the tumorigenesis of EFTs. We have previously reported that cyclin E expression was upregulated in EFT cells and in EWS-Fli1 transformed fibroblastic cells. However, the mechanism of the overexpression of cyclin E by EWS-Fli1 is still unknown. In our study, we investigated the mechanism of transactivation of the cyclin E gene in EFT cells. We found that EWS-Fli1 enhanced the activity of the cyclin E gene promoter partially through E2F binding sites in the promoter. In addition, the basic transcriptional factor, Sp1, might also be involved in the transactivation of the cyclin E gene by EWS-Fli1. To study the biological significance of cyclin E overexpression in EFT cells, we used flavopiridol, a pan-cyclin-dependent kinase (CDK) inhibitor and found that flavopiridol efficiently suppressed the growth of EFT cells in vitro and in vivo by the inhibition of cyclinE/CDK2 kinase activity and the induction of apoptosis. These results suggest that targeting of the cyclin/CDK complex may provide new insight into treatment of EFTs.

Antitumor effects of histone deacetylase inhibitor on Ewing's family tumors

A chimeric protein, EWS-Fli1, identified in most Ewing's family tumors (EFTs) has been shown to be associated with the tumorigenicity of EFTs. We have previously reported that p21(Waf1/Cip1) expression was inhibited by EWS-Fli1 in EFTs. Histone deacetylase inhibitors (HDACIs) are known to up-regulate p21(Waf1/Cip1) expression in various cells and show promise as a cancer therapy. Here, we demonstrate the possible involvement of EWS-Fli1 in the activities of both histone acetylation and deacetylation, as well as the potential use of HDACIs as an antitumor agent for EFTs. A novel HDACI, FK228, strongly induced p21(Waf1/Cip1) expression, leading to the hypophosphorylation of retinoblastoma protein (Rb) in EFT cells. Results indicated that EWS-Fli1 deregulated histone acetylation through both the repression of histone acetyltransferase (HAT) and the enhancement of histone deacetylase (HDAC) activities in EFT cells. FK228 treatment blocked both of the abnormal functions of EWS-Fli1. Expressions of EWS-Fli1 protein and mRNA were also inhibited by HDACIs. We suggest that HDACIs might inhibit the expression of EWS-Fli1 via the suppression of the EWS promoter activity. FK228 demonstrated potent growth inhibitory effects on EFT cells at nanomolar concentrations, as well as an apparent distinction in the apoptotic effects between EFT and normal cells. Moreover, intraperitoneal administration of FK228 significantly inhibited tumor growth and induced apoptosis in EFTs in vivo. These results suggest that HDACI might be a promising reagent for use in molecular-based chemotherapy against EFTs.

IDENTIFICATION OF AN IMMUNOGENIC EWS-FLI1-DERIVED HLA-DR-RESTRICTED T HELPER CELL EPITOPE

Immunotherapy with tumor-specific peptide-loaded dendritic cells represents a promising therapeutic approach for patients with multifocal primary or early relapsed Ewing family tumors (EFT). The authors therefore screened a peptide library derived from the fusion region of the EFT-specific chimeric transcription factor EWS-FLI1 for immunogenic peptides. T-cell priming with 10 peptides was evaluated using IFNγ video-assisted automated enzyme-linked immunospot technique. The authors report the identification of the first EFT-specific immunogenic T-cell epitope so far. Its identification will lead to a better understanding of EFT immunology and may improve DC-based immunotherapy.

Tyrosine Kinase Receptor Inhibition with Ecteinascidin-743 for the Ewing Family of Tumors

Tyrosine Kinase Receptor Inhibition with Ecteinascidin-743 for the Ewing Family of Tumors

Concomitant Ewing sarcoma and acute lymphoblastic leukemia in a 5-year-old girl

Malignancies from the Ewing family of tumors and acute lymphoblastic leukemia (ALL) are not known to be associated with each other. A 5-year-old girl was incidentally found to suffer from acute lymphoblastic leukemia during bone marrow staging for Ewing sarcoma of the radius. The simultaneous presence of two distinct neoplasms was confirmed by RT-PCR, with EWS/FLI1 type 1 rearrangement in the bone tumor and TEL/AML1 rearrangement in the marrow. She was treated with chemotherapy, radiotherapy, and surgery and was in remission of both diseases 31 months after diagnosis.

Advances that are changing the diagnosis and treatment of malignant bone tumors

Several neoplastic conditions may affect bone. These include primary bone tumors as well as metastatic disease from distant primary sites. Often, these entities produce symptoms that may be difficult to distinguish from those of various rheumatologic entities. The purpose of this review is to discuss recent developments in orthopedic oncology, with special attention given to advances that are changing the diagnosis and treatment of bone sarcomas and carcinomas metastatic to bone. Much effort in the field of musculoskeletal oncology has been dedicated to the elucidation of the molecular mechanisms underlying bone sarcomas, especially in the case of osteogenic sarcoma and Ewing family tumor. Telomere maintenance mechanisms are emerging as potential targets for anticancer therapy. The most exciting advances have been in the development of novel treatments for cancers affecting bone. The anticancer effects of bisphosphonates, cyclooxygenase-2 inhibitors, and statins may expand their indications to the treatment of primary bone tumors. Finally, new expandable implants have been developed for the treatment of bone tumors in growing children. These devices may help solve some of the problems encountered with reconstruction of the growing skeleton. Recent discoveries in the molecular mechanisms of bone sarcomas may help to elucidate the pathogenesis of these rare diseases. This, combined with the recent findings of the anticancer effects of bisphosphonates, cyclooxygenase-2 inhibitors, and statins, may lead to the development of novel treatments for sarcomas of bone and of carcinomas metastatic to bone.

Discriminate gene lists derived from cDNA microarray profiles of limited samples permit distinguishing mesenchymal neoplasia ex vivo

Mesenchymal neoplasia comprises a heterogeneous group of tumors with over 200 benign neoplasms and 100 sarcomas. Currently, tumors are classified using histologic and immunocytologic characteristics, with diagnostic error rates reported as high as 40% of cases. As a feasibility study, our goal was to generate a preliminary discriminatory gene list for selected mesenchymal tumors, including sarcomas. This technique may enable an eventual molecular classification schema based on expression profiles that can complement current clinical and pathologic diagnostic procedures in mesenchymal tumors. cDNA microarray analyses were preformed on connective tissue tumors obtained at time of surgical resection or biopsy. Messenger RNA (mRNA) from four general tumor classes was competitively hybridized against a human dermal fibroblast cell line comparator and the resulting gene expression profiles processed by ANOVA and linear discriminate analysis. The tissue classification involved 18 patients with malignant peripheral nerve sheath tumors, giant cell containing tumors, benign spindle cell lesions, or Ewing's family of tumors. Lymph nodes from two patients served comparative purposes. Twenty-five differentially regulated genes considered most variable among the five tissue classes were identified. The tissues were segregated into five classes by linear discriminate analysis. Linear discriminate analysis of cDNA gene expression profiles partitioned mesenchymal tumor classes, even when constrained by limited sample sizes.

Primary multidisciplinary management of extremity soft tissue sarcomas

Soft tissue sarcomas (STS) are a rare and heterogeneous group of malignancies that most commonly present as large painless masses deep in the muscular compartments of the extremities. Investigation and treatment of these patients must be undertaken at a tertiary referral unit. Staging studies must include a high-quality magnetic resonance imaging (MRI) scan of the local site and a computed tomography (CT) scan of the chest to investigate for possible metastatic disease. Review of biopsy material must be undertaken by an experienced musculoskeletal pathologist. Currently, histologic diagnosis and grade are assigned to the tumor, but in tumors such as synovial sarcoma and Ewing's family of tumors, molecular evaluation is becoming crucial for diagnostic, prognostic, and therapeutic reasons. Surgical resection of sarcomas with negative surgical margins remains the mainstay of treatment. Surgical treatment alone is indicated for small superficial masses that are not adjacent to bone or neurovascular structures. However, in large deep masses where surgical margins are likely to be close in order to preserve important neurovascular structures and bone, combined treatment using surgical resection and radiation results in acceptable local control rates and reasonable patient function. It is incumbent on the management team to decide on timing of radiotherapy as a practice. This decision is based on several factors but potential complications must be taken into account. In this regard the long-term effects on normal tissues must be considered. Functional outcome has become an essential consideration when recommending treatment. If promising techniques become available to reduce the incidence of wound complications or to decrease the dose of radiation to normal tissue, preoperative radiation should be considered in light of its lower incidence of long-term effects that result in reduction in patient function.

DNA Microarrays Reveal Relationship of Ewing Family Tumors to Both Endothelial and Fetal Neural Crest-Derived Cells and Define Novel Targets

Ewing family tumors (EFTs) are small round blue cell tumors that show features of neuroectodermal differentiation. However, the histogenetic origin of EFTs is still a matter of debate. We used high-density DNA microarrays for the identification of EFT-specific gene expression profiles in comparison with normal tissues of diverse origin. We identified 37 genes that are up-regulated in EFTs compared with normal tissues and validated expression of these genes in EFTs by both conventional and quantitative reverse transcription-polymerase chain reaction. The expression pattern of EFT-associated genes in normal tissues indicated a high similarity between EFTs and fetal and neuronal as well as endothelial tissues and supports the concept that a primitive neural crest-derived progenitor at the transition to mesenchymal and endothelial differentiation is transformed in EFTs. EFT-associated genes could be used for molecular discrimination between EFTs and other small round blue cell tumors and clearly identified a cell line (SK-N-MC) that was initially established as neuroblastoma as being an EFT. Ectopic expression of the EFT-specific EWS-FLI1 fusion protein in human embryonic kidney (HEK293) cells was not sufficient to induce the complete EFT-specific gene expression signature, suggesting that the EFT-specific gene expression profile is not just a consequence of EWS-FLI1 expression but depends on the histogenetic background of the EFT stem cell.

Ewing's Sarcoma of the Axial System in Patients Older Than 15 Years: Dismal Prognosis Despite Intensive Multiagent Chemotherapy and Aggressive Local Treatment

Older age and axial location of Ewing's sarcoma have been reported as unfavorable prognostic factors. The records of patients older than 15 years with the Ewing's family of tumors were reviewed retrospectively. After the induction chemotherapy consisting of alternating vincristine, adriablastin, cyclophosphamide (VAC) and etoposide, ifosfamide with mesna protection (IE), a local treatment modality was chosen based on tumor and patient characteristics. Twenty-five patients with a median age of 19 years were evaluated. Median follow-up was 26 months (range 4-58). Seventeen patients (68%) had died. In univariate analysis, factors predictive of shorter survival were the patients presenting with metastatic disease, with the primary tumor located at the pelvis, those who never achieved complete response to chemotherapy and those who had chemotherapy for <12 months. Only a negative link with pelvic location was observed in multivariate analysis [risk ratio 7.5; 95% confidence interval (CI) 1.52-37.06; P = 0.0134]. Median progression-free survival (PFS) and overall survival (OS) were 10 months (95% CI 6.2-13.8) and 14 months (95% CI 9.3-18.7), respectively. Cumulative 2-year PFS and OS were 19.0% (95% CI, SD +/-8.4) and 32.7% (95% CI, SD +/-9.8), respectively. The prognosis of patients with axial Ewing's sarcoma is dismal despite an intensive, multimodality approach including multiagent, alternating chemotherapy, surgery and/or radiotherapy. A more aggressive approach should be considered for this group of Ewing's sarcoma patients.