EWS Exon Research Articles

Abstract 154: Establishment and characterization of a novel clear cell sarcoma organoid model derived from a human biopsy

Abstract Clear Cell Sarcoma (CCS) is an extremely aggressive, rare soft tissue cancer accounting for approximately 1% of all sarcomas. Often diagnosed in young adults with a median age of 25 years, CCS has a strong propensity for local recurrence and metastasis with an overall high mortality rate. Current treatment recommendations include radical surgical extirpation, followed by radiation or chemotherapy. Nonetheless, there is a need for extensive postoperative follow-up as the efficiency of adjuvant therapy in managing this disease remains poor. Consequently, it is imperative to identify clinically relevant models to advance our understanding of CCS tumor biology and facilitate the development of novel therapies. Compared to conventional preclinical tumor models, patient-derived organoids (PDOrgs) better recapitulate the patient tumor biology, genetic heterogeneity, and therapeutic responses. Here, we describe the development and validation of a human biopsy-derived CCS organoid model 1010561-T. 1010561-T was established from a male patient's metastatic paraspinal mass. This PDOrg demonstrates a moderate growth rate (doubling time of ~20 days) and pleomorphic traits, consisting mainly of solid, grape-like clusters, as well as elongated cells of mixed morphology. Furthermore, this model was successfully maintained over 7 months in culture and remained stable more than one year after cryopreservation. 1010561-T exhibits the cytogenetic hallmark of CCS which is the presence of a recurrent t(12;22)(q13;q12) chromosomal translocation, resulting in a fusion between the Ewing sarcoma gene and activating transcription factor 1 (EWS/ATF1 fusion). Reverse-transcription polymerase chain reaction (RT-PCR) confirmed that 1010561-T harbors a dual EWS/ATF1 fusion, namely EWS exon 8 to ATF1 codon 65 fusion (type 1) and EWS exon 7 to ATF1 codon 110 fusion (type 3). Quantitative RT-PCR and immunocytochemistry revealed strong expression of melanocytic differentiation markers typical of CCS including melanocyte inducing transcription factor (MITF), melan-A (MLANA), premelanosome protein (PMEL), and S100 calcium binding protein A11 (S100A11). Additionally, the gene expression profile of 1010561-T is in concordance with a well-established CCS cell line, SU-CCS-1. To our knowledge, 1010561-T is the first human CCS organoid model. It is, therefore, a valuable resource for advancing our understanding of the biology of this very rare disease, in addition to being a useful tool for validating new preclinical therapies that are undoubtedly needed to achieve better clinical outcomes for patients. To this end, 1010561-T will be transferred to the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov) where it will be made available to the scientific community. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201800001I. Citation Format: Petreena S. Campbell, Erik D. Harris, Nancy Moore, Ralph E. Parchment, Nathan P. Coussens, Beverly A. Teicher, Alice Chen, James H. Doroshow, Annamaria Rapisarda. Establishment and characterization of a novel clear cell sarcoma organoid model derived from a human biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 154.

Diagnostic utility of EWS break-apart fluorescence in situ hybridization in distinguishing between non-cutaneous melanoma and clear cell sarcoma

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma with morphological similarities to malignant melanoma (MM), but with a distinct genetic background that includes the chromosomal translocation t(12;22)(q13;q12). Clear cell sarcoma is often misdiagnosed as MM because of similarities in target locations and immunophenotypes. Eighteen cases with MM in non-cutaneous sites were subjected to fluorescence in situ hybridization (FISH) to assess EWS gene breakage. Tissue microarrays were constructed using formalin-fixed, paraffin-embedded tissue and the EWSR1 (22q12) dual-color, break-apart rearrangement probe (Vysis) was used. Two patients were classified as CCS with EWS gene rearrangement, with a mean of 67.5% positive cells per sample according to break-apart FISH. The remaining 16 patients lacked break-apart signals of the EWS gene. The presence of type 1 (EWS exon 8-ATF1 exon 4) fusion transcripts was confirmed in FISH-positive patients by RT-PCR. Retrospective analysis revealed that the masses were located in the foot and buttock, respectively. Morphologically, tumor cells were not typical for those of CCS or MM. Break-apart FISH is an accurate and convenient method for differentiating between MM and CCS. Molecular detection of EWS gene rearrangement, either by break-apart FISH or RT-PCR, is mandatory in subjects with melanotic tumors of soft tissue.

Superficial primitive neuroectodermal tumor/Ewing sarcoma (PN/ES): same tumor as deep PN/ES or new entity?

Primitive neuroectodermal tumor/Ewing sarcoma (PN/ES) is a single clinical, morphologic, and molecular small round cell tumor entity. These are generally found in deep soft tissue or bone of young male patients, with poor behavior. Location in dermis is unexpected; only rare cases are reported. Cases coded as "dermal," "cutaneous," or "skin" PN/ES were retrieved from our consultation files. Only primary dermal cases were included. Those otherwise diagnosed or with incomplete material were excluded. There were 13 dermal PN/ES cases, consisting of 10 women and 3 men. Ages ranged from 2 to 67 years (mean, 28 years). Locations included groin or thigh (4), back or shoulder (3), neck (1), chest (1), scalp (1), forehead (1), hand (1), and foot (1). Most cases were small (0.5-2.3 centimeters) and painful, and persisted for less than 1 year. All were located within the dermis. Three caused pedunculation; 9 also involved superficial subcutis. All but 1 of the metastasizing tumors were round and encapsulated. All were composed of round to oval cells with a vague rosetting pattern, slightly overlapping nuclei, finely stippled chromatin, clear to eosinophilic cytoplasm, and collagenous stroma. Median mitotic activity was 8 per 10 high-power fields. Necrosis was present in three cases. All had globular periodic acid Schiff positivity and distinctive cytoplasmic membrane CD99 reactivity. One case studied was positive for Fli-1. All were negative for leukocyte common antigen, Tdt, CD3, CD20, CD79, CK20, pankeratin, epithelial membrane antigen, chromogranin, neurofilament protein, carcinoembryonic antigen, desmin, actin, diffuse S100 protein, and HMB45. Nine cases with material for reverse transcription-polymerase chain reaction revealed 1 positive type 2 translocation (EWS exon 7 to Fli-1 exon 5), 4 negative, and 4 "unable to amplify." Treatment was by wide excision; 9 received chemotherapy and 6 radiation. Follow-up of 11 (85%) cases revealed the following: 1 metastasis to stomach and death at 3 years; 10 years without disease (median, 9.0 years). Cutaneous PN/ES is a superficial round cell tumor in older women, with better prognosis than deep PN/ES. These may have a hitherto unrecognized variant genetic abnormality.

Desmoplastic small cell tumor of soft tissue: Molecular variant of EWS‐WT1 chimeric fusion

A 7-year-old girl was hospitalized because of a tumorous mass in her left periorbital region. The tumor was removed by local excision. The soft-part tumor recurred in the parotid gland region 4 months later, and a second recurrence was noted on the left side of the neck 3 years and 3 months thereafter. The patient had not received chemotherapy or local irradiation. Histological and immunohistochemical examinations of the recurrent masses revealed morphological characteristics of small cell proliferation with desmoplastic stroma that were similar to those of the initial tumor. The cellular components showed immunoreactivity for desmin, cytokeratin, vimentin, and epithelial membrane antigen in part, but the cells were negative for myogenin, CD99, and neuron-specific enolase. These findings suggested a diagnosis of desmoplastic small cell tumor, despite its extra-abdominal location. The histological diagnosis was confirmed by reverse transcriptase polymerase chain reaction, which demonstrated an EWS-WT1 chimeric fusion gene. An in-frame fusion of EWS exon 9 and WT1 exon 8 was subsequently identified by cloning and sequencing. The chimeric fusion gene might be related to the tissue-specific phenotype of desmoplastic small cell tumors, although further investigation of this speculation is necessary.

Visceral Clear Cell Sarcoma of Soft Tissue with Confirmation by EWS-ATF1 Fusion Detection

Clear cell sarcoma of soft tissue (CCS-ST) is a rare malignant neoplasm characterized by a tumor-defining translocation [t(12;22) (q13;q12)], resulting in the EWS-ATF1 gene fusion. An extremely limited number of visceral CCS-ST cases have been reported in the literature. Here the authors report a visceral CCS-ST in a Hispanic adolescent male with a large infiltra-tive mass involving the small bowel. The tumor was evaluated by light microscopy, immunocytochemistry, electron microscopy, cytogenetics, and molecular genetics. The tumor cells were strongly positive for S-100 protein, but negative for HMB-45. Rare premelanosomes were identified only after an extensive search with electron microscopy. Cytogenetics showed a characteristic t(12;22)(q13;q12) for CCS-ST with isochromosome 18q and trisomy 22. An EWS exon 8 sense primer and an antisense ATF1 primer were employed for detection of the CCS-ST tumor-defining EWS-ATF1 translocation, using reverse transcriptase–polymerase chain reaction techniques (RT-PCR), and the fusion gene breakpoint underwent DNA sequencing. This tumor is exceptional, because it is the first visceral CCS-ST that has been confirmed by RT-PCR and DNA sequencing. This case also illustrates the necessity of a multimodal approach to tumor diagnosis, and the utility of cytogenetics and molecular pathology in confirming the diagnosis of CCS-ST and eliminating conventional metastatic or primary visceral malignant melanoma as a consideration.

Identification of Various Exon Combinations of theews/fli1Translocation: An Optimized RT-PCR Method for Paraffin Embedded Tissue

Chromosomal translocations t(11;22) (q24;q12) are characteristic of about 80-90 % of Ewing's sarcoma family of tumors [bone and soft tissue Ewing's sarcoma and peripheral neuroectodermal tumors (PNET)]. They generate ews/fli1 rearrangements showing great diversity in breakpoint exon combination. In about 5 % of Ewing's tumors, ews is fused to the erg gene at 21q22. The various chimeric proteins encoded may function as aberrant oncogenic transcription factors. These specific translocations can be used for exact molecular diagnosis in these poorly differentiated small round-cell tumors. Moreover, the prognostic relevance of different translocational variants has been previously suggested. Furthermore, the sensitive molecular detection of minimal metastatic and residual disease and its clinical significance can be evaluated. To address these questions more definitively in the large number of patients registered in multicenter studies, it is often necessary to access archival paraffin-embedded tumor tissue if no fresh or frozen tumor material is available for analysis by RT (reverse transcription)-PCR. Specific problems arise from formalin-fixed and paraffin-embedded tissue due to the degradation of RNA and insufficient extraction efficiency. Therefore, primer distance and product size are limited for successful PCR amplification. This conflicts with the requirement for identification of various possible exon combinations by PCR simultaneously using one single primer pair with larger distance.We examined paraffin embedded soft part tumor tissue samples from 47 Ewing's tumor patients. Patients were treated according to either CWS (Cooperative Weichteilsarkomstudie, CWS-91 or CWS-96) or Euro-E.W.I.N.G. 99 therapy protocols.We established a novel RT-PCR method, using 3 different exon specific sets of PCR primer pairs, selected according to the coding ews and fli1 nucleotide sequences (NCBI database), suitable for RT-PCR identification of variant ews/fli1 fusion transcripts in RNA isolated from formalin-fixed, paraffin-embedded tissue. For use in combination with ews -primer, an erg specific primer was selected to alternatively test for ews/erg fusion transcripts. As positive control for the integrity of isolated mRNA, we used the ubiquitously expressed gapdh transcript for RT-PCR amplification in each sample.In 31 cases (= 66 %) of 47 paraffin samples of Ewing's tumors analysed, gapdh control indicated adequate quality of RNA. In 16 cases no gapdh control fragment was amplifiable, nevertheless in 2 of these 16 samples distinct ews fusion products could be detected. In 23 cases we identified ews fusion transcripts. Thereof in 65 % ews exon 7 being fused to fli1 exon 6 (fusion type I), in 22 % to fli1 exon 5 (fusion type II). In 4 % each ews exon 10 being juxtaposed to fli1 either exon 6 or exon 5, respectively. An ews/erg fusion was detected in 4 % ( ews exon 7 fused to erg exon 6). In 10 samples, a gapdh fragment was amplified, but no ews/fli1 or - erg fusion transcript could be identified. The reference pathological review (I. L., Kiel, Germany) disproved the primary histopathology in 5 cases.Using our different sets of exon specific primer pairs, it was possible to detect 4 different breakpoints of ews/fli1 fusion transcripts and the ews/erg fusion by RT-PCR in RNA isolates from formalin-fixed, paraffin-embedded Ewing's tumor tissue. This method can be a very useful alternative in clinical situations (to ensure diagnosis and perform minimal metastatic and residual disease investigations) and in order to assess prognostic significance of translocation subtypes when no fresh tumor tissue is available.

Population-based genetic alterations in Ewing’s tumors from Japanese and European Caucasian patients

BackgroundThe incidence of Ewing’s tumors (ETs) is lower in Asians or African-Americans than in Caucasians. Patients and methodsJapanese ETs were available for analysis of chromosomal aberrations by comparative genomic hybridization (n = 16) and for expression of chimeric EWS transcripts by reverse-transcriptase polymerase chain reaction (n = 11). These results in Japanese patients were compared with those of 62 ETs in European Caucasian patients registered in the European Intergroup Cooperative Ewing’s Sarcoma Study. ResultsJapanese patients with ET had lower overall survival (P = 0.0446) and relapse-free survival (P = 0.0371) compared with European Caucasian patients. Ten of 11 Japanese ETs and 31 of 62 European Caucasian ETs had type I (EWS exon 7 to FLI1 exon 6) fusion transcripts. In Japanese ETs, the median numbers of chromosomal aberrations were 2.0 and 6.0 in 11 primary tumors and five relapsed tumors, respectively. In European Caucasian ETs, the median number of changes were 2.5 and 5.0 in 52 primary and 10 relapsed tumors, respectively. Frequent gains were 8q (38%), 8p (31%) and 12q (25%) in Japanese ETs and 8q (52%), 8p (48%) and 12q (19%) in European Caucasian ETs. Frequent losses were 19q (44%), 19p (38%) and 17p (25%) in Japanese ETs and 16q (21%), 19q (18%) and 17p (15%) in European Caucasian ETs. The incidence of losses of 19p (P = 0.0215) and 19q (P = 0.0277) were significantly higher in Japanese ETs than in European Caucasian ETs. An amplification(1p33-p34) was observed in only one Japanese ET. ConclusionsJapanese patients with ET in this study had a worse prognosis than European Caucasian patients. In molecular genetic analyses, Japanese ETs had a higher frequency of loss of chromosome19 than European Caucasian ETs. Different genetic aberrations may explain the different incidences and prognoses of ET between Caucasian and Japanese patients.

Molecular genetic characterization of the EWS/CHN and RBP56/CHN fusion genes in extraskeletal myxoid chondrosarcoma.

Extraskeletal myxoid chondrosarcoma (EMC) is a soft-tissue neoplasm cytogenetically characterized by the translocations t(9;22)(q22;q11-12) or t(9;17)(q22;q11), generating EWS/CHN or RBP56/CHN fusion genes, respectively. In the present study, 18 EMCs were studied both cytogenetically and at the molecular level. Chromosomal aberrations were detected in 16 samples: 13 with involvement of 9q22 and 22q11-12, and three with rearrangements of 9q22 and 17q11. Fifteen cases had an EWS/CHN fusion transcript and three had an RBP56/CHN transcript. The most frequent EWS/CHN transcript (type 1; 10 tumors), involved fusion of EWS exon 12 with CHN exon 3, and the second most common (type 5; two cases) was fusion of EWS exon 13 with CHN exon 3. In all tumors with RBP56/CHN fusion, exon 6 of RBP56 was fused to exon 3 of CHN. By genomic XL PCR and sequence analyses, the breakpoints from 14 cases were mapped in the EWS, RBP56, and CHN genes. In CHN, 12 breakpoints were found in intron 2 and only two in intron 1. In EWS, the breaks occurred in introns 7 (one break), 12 (eight breaks), and 13 (one break), and in RBP56 in intron 6. Repetitive elements such as Alu and LINE sequences seem to have limited, if any, importance in the genesis of EWS/CHN and RBP56/CHN chimeras. Furthermore, there were no chi, chi-like, topoisomerase II, or translin consensus sequences in the introns harboring the translocation breakpoints, nor could the number of topo I sites in EWS, RBP56, and CHN introns explain the uneven distribution of the breakpoints among EWS or CHN introns. Additional genetic events, such as nucleotide insertions, homologies at the junction, deletions, duplications, and inversions, were found to accompany the translocations, indicating that the chromosomal translocations do not require sequence-specific recombinases or extensive homology between the recombined sequences.

Molecular genetic characterization of the EWS/ATF1 fusion gene in clear cell sarcoma of tendons and aponeuroses.

Clear cell sarcoma (CCS) is a rare malignant soft tissue tumor particularly associated with tendons and aponeuroses. The cytogenetic hallmark is the translocation t(12;22)(q13;q12) resulting in a chimeric EWS/ATF1 gene in which the 3'-terminal part of EWS at 22q is replaced by the 3'-terminal part of ATF1 at 12q. To date, only 13 cases of CCS have been analyzed for fusion genes at the transcription level, and there is no information about the breakpoints at the genomic level. In the present study, we describe the molecular genetic characteristics of CCS from 10 patients. Karyotypes were obtained from 10 cases, 7 of which showed the characteristic t(12;22). As an initial step in the characterization of the EWS/ATF1 and ATF1/EWS chimeras, we constructed an exon/intron map of the ATF1 gene. The entire ATF1 gene spanned >40 kb and was composed of 7 exons. Intron 3, in which most of the genomic breakpoints occurred, was to a large extent (83%) composed of repetitive elements. RT-PCR amplified EWS/ATF1 cDNA fragments in all patients and ATF1/EWS cDNA fragments in 6 of 10 patients. Four types of EWS/ATF1 chimeric transcript, designated types 1-4, were identified. The most frequent chimeric transcript (type 1) was an in-frame fusion of exon 8 of EWS with exon 4 of ATF1. This was the only chimeric transcript in 5 patients but found together with other variants in 3 tumors. The type 2 transcript of EWS/ATF1, an in-frame fusion of exon 7 of EWS with exon 5 of ATF1, was detected in 4 patients, as the only transcript in 1 case and together with other variants in 3 cases. An in-frame fusion of exon 10 of EWS with exon 5 of ATF1 (type 3) was found in 1 patient as the only transcript, and an out-of-frame fusion of EWS exon 7 with ATF1 exon 7 (type 4) was detected in 1 patient together with type 1 and type 2 transcripts. Sequencing of the amplified ATF1/EWS cDNA fragments showed in 5 patients that ATF1 exon 3 was fused with EWS exon 10, resulting in an out-of-frame chimeric transcript. In 1 case, nt 428 of ATF1 (exon 4) was fused with EWS exon 8; at the junction, there was an insertion of 4 nucleotides, also resulting in an out-of-frame transcript. Genomic extra long PCR and sequence analysis mapped the genomic breakpoints to introns 7, 8 and 9 of EWS and intron 3 and exon 4 of ATF1. While a simple end-to-end fusion was observed in 2 cases, additional nucleotides were found at the junctions in 2 other cases. In addition, topoisomerase I consensus sequences were found close to the junctions, suggesting that this enzyme may participate in the genesis of the EWS/ATF1 fusion.

Transcriptional regulation of IGF-I receptor gene expression by novel isoforms of the EWS-WT1 fusion protein.

The EWS family of genes is involved in numerous chromosomal translocations that are characteristic of a variety of sarcomas. A recently described member of this group is desmoplastic small round cell tumor (DSRCT), which is characterized by a recurrent t(11;22)(p13;q12) translocation that fuses the 5' exons of the EWS gene to the 3' exons of the WT1 gene. The originally described chimera comprises exons 1-7 of EWS and exons 8-10 of WT1. We have previously reported that the WT1 protein represses the expression of the IGF-I receptor gene, whereas the EWS(1-7)-WT1(8-10) fusion protein activates IGF-I receptor gene expression. It has recently become apparent that EWS-WT1 chimeras produced in DSCRT are heterogeneous as a result of fusions of different regions of the EWS gene to the WT1 gene. We have recently characterized additional EWS-WT1 translocations that involve the juxtaposition of EWS exons 7 or 8 to WT1 exon 8, and an EWS-WT1 chimera that lacks EWS exon 6. The chimeric transcription factors encoded by these various translocations differ in their DNA-binding characteristics and their ability to transactivate the IGF-I receptor promoter. These data suggest that the molecular pathology of DSRCT is more complex than previously appreciated, and that this diversity may provide the foundation for predictive genotype-phenotype correlations in the future.

Molecular Diagnosis of Clear Cell Sarcoma: Detection of EWS-ATF1 and MITF-M Transcripts and Histopathological and Ultrastructural Analysis of 12 Cases

Clear cell sarcoma (CCS), also known as melanoma of soft parts, is an uncommon deep soft tissue tumor presenting typically in the lower extremities of young adults. Previous cytogenetic studies have established the specificity of the recurrent t(12;22)(q13;q12), resulting in a EWS-ATF1 fusion, for CCS. The prevalence of the EWS-ATF1 fusion in CCS remains unclear, since most genetically confirmed CCS have been reported as isolated cytogenetic or molecular diagnostic case reports. We therefore studied histologically confirmed CCS from 12 patients for the presence of EWS-ATF1 by reverse-transcriptase polymerase chain reaction (RT-PCR), using RNA extracted from either frozen (four cases) or formalin-fixed paraffin-embedded (eight cases) material. All primary tumors were located in the deep soft tissues of the extremities. Histologically, 10 cases had a typical epithelioid nested appearance. Most or all cases showed immunostaining for HMB45 (12 of 12), S-100 protein (10 of 12), and MITF (12 of 12). Ultrastructural analysis showed melanosomes in six of seven cases. The presence of an EWS-ATF1 fusion transcript was identified by RT-PCR in 11 of 12 cases (91%), all of which showed the same fusion transcript structure, namely the previously described in-frame fusion of EWS exon 8 to ATF1 codon 65. RT-PCR analysis for the melanocyte-specific splice form of the MITF transcript was positive in all cases tested (4 of 4). These data confirm that EWS-ATF1 detection can be used as a highly sensitive diagnostic test for CCS and that CCS expresses the melanocyte-specific form of the MITF transcript, further supporting its genuine melanocytic differentiation.

Primitive Neuroectodermal Tumors of the Biliary and Gastrointestinal Tracts: Clinicopathologic and Molecular Diagnostic Study of Two Cases

Primitive neuroectodermal tumor (PNET) is a prototypic malignant small round cell tumor of childhood that is characterized in most cases by t(11;22) resulting in an EWS-FLI1 gene fusion. Once thought to be uncommon, PNET now accounts for almost 20% of malignant soft tissue tumors in children. Increased recognition of PNET is partly due to advances in immunohistochemistry and molecular diagnostics, which have led to the identification of the tumor in non-classical sites. We report the clinical, histologic, immunohistochemical, and molecular findings of two visceral PNETs of the digestive system--one involving the small intestine and the other involving the hepatic duct. Histologically, each tumor was composed of malignant small cells growing in sheets, nests, and lobules; the tumor cells of both cases showed characteristic immunoreactivity for vimentin and O13 (CD99). Reverse transcription-polymerase chain reaction (RT-PCR) analysis for t(11;22) using nested primers was performed with RNA extracted from paraffin-embedded, formalin-fixed tissue and demonstrated an EWS exon 7 to FLI1 exon 5 fusion in both cases, confirmed by Southern blot hybridization and DNA sequence analysis. These results illustrate the expanded clinicopathologic profile of PNET, and demonstrate that visceral PNETs, despite their unusual sites of presentation, maintain the characteristic immunohistochemical and genetic features of PNETs at more conventional sites.

Molecular characterization of the genomic breakpoint junction in a t(11;22) translocation in Ewing sarcoma

Polymerase chain reaction (PCR)-based nucleotide sequence analysis was performed in 12 cases of Ewing sarcoma on the cDNA and/or genomic DNA breakpoint regions of a t(11;22)(q24;q12), which joins the EWS gene located on chromosome 22 with the FLI1 gene located on chromosome 11, in order to understand the molecular mechanism of this translocation. Reverse transcriptase-PCR on total tumor cell RNA from the examined cases showed five types of EWS-FLI1 chimeric product, resulting from various junctions between EWS exon 7 or 10 with FLI1 exon 5, 6, or 8. Sequencing of the genomic fusion junctions of EWS-FLI1 in seven cases showing three types of the chimeric cDNA products revealed that most of the breakpoint junctions shared common nucleotide(s) from both genes, and that the breakpoints in EWS introns 7 and 10 clustered within 100 bp and 300 bp, respectively. All the junctions were found to be flanked by various oligomers, among which a consensus sequence, 5'-AGAAAARDRR-3', was found near the breakpoints of both genes in four cases, suggesting that these oligomers may have a functional significance in the genesis of t(11;22). In addition to these oligomers, sequences highly homologous to Alu repeats and/or eukaryotic topoisomerase II cleavage sites were located near, or flanked, or even encompassed, the breakpoints in most of the cases examined. Thus, these sequences may also mediate DNA double-strand breakage and rejoining to generate the t(11;22). Genomic sequence analysis of both EWS-FLI1 and FLI1-EWS chimeric genes in three of the seven cases demonstrated a deletion and duplication of both EWS and FLI1 sequences in two cases and no gain or loss in one case. The present findings suggest that multiple mechanisms may be operative for the break and rejoining of the fragments of chromosomes 11 and 22 in the genesis of t(11;22), and that some of these translocations are asymmetric at the molecular level.

Novel Breakpoints of the EWS Gene and the WT1 Gene in a Desmoplastic Small Round Cell Tumor

We report here a 15-year-old boy with an intraabdominal desmoplastic small round cell tumor (DSRCT). Cytogenetic analysis of the tumor cells showed the chromosomal translocation (11;22). Reverse-transcriptase polymerase chain reaction and sequencing analysis revealed a chimeric transcriptional message of the EWS gene exon 10 fused to the WT1 gene exon 8. The typical chimeric transcript seen in DSRCT is an in-frame fusion of EWS exon 7 to WT1 exon 8. The tumor in this case had a novel and longer chimeric transcript, which should be a potent transcription factor. Genetic analysis is a very powerful and specific aid in the differential diagnosis of small round cell tumors.

Does expression of different EWS chimeric transcripts define clinically distinct risk groups of Ewing tumor patients?

Because of the high heterogeneity of EWS gene fusions with FLI1 and ERG genes due to variable chromosomal breakpoint locations in Ewing tumors (ET) (14 different chimeric transcripts identified so far), we evaluated the clinical impact of the expression of diverse fusion transcripts in ET patients. In a European multicenter study, 147 ET were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and the molecular data statistically compared with all clinical data available. Most tumors expressed chimeric transcripts with fusion of EWS exon 7 to FLI1 exon 6 (75 of 147) (type I) or five (39 of 147) and EWS exon 10 to FLI1 exon 5 (eight of 147) or 6 (five of 147). In five cases, chimerism between EWS exon 9 and FLI1 exons 4 and EWS exon 7 and FLI1 exon 7 or 8 was observed. Fifteen cases of EWS-ERG rearrangement were identified. In 85 of these patients treated in the European Cooperative Ewing Sarcoma Studies, molecular results were analyzed in comparison to age, sex, tumor localization, tumor volume, and disease extension. No significant correlation between the various fusion types and these features were observed. Relapse-free survival (RFS) for the 31 patients with localized disease and fusion type I tended to be longer compared with the 24 patients with localized tumors bearing other chimeric transcripts (P = .04). Results suggest a possible advantage in PFS for patients with localized disease and fusion type I transcripts, although this will require prospective validation with a larger number of patients and longer follow-up periods.

Molecular analysis of a t(II;22) translocation junction in a case of Ewing's sarcoma

Polymerase chain reaction (PCR)-directed sequence analysis was performed to characterize the genomic and cDNA breakpoint junctions of t(11;22) (q24;q12) translocation in a case of Ewing's sarcoma, in which the EWS gene located on chromosome 22 is rearranged with the FL11 gene located on chromosome 11. RNA-PCR revealed the novel chimeric product of EWS/FL11 gene on the derivative chromosome (der) 22, resulting from a probable fusion of EWS exon 7 to FL11 exon 9. Sequencing of the PCR-amplified genomic fragments of the fusion genes showed that the breakpoints on der(22) occurred in EWS intron 7 and, most probably, in FL11 intron 8. Those of the untranscribed counterpart on der(11) were located in the same FL11 intron and in EWS exon 11, with deletion of a considerable amount of sequences from both genes. These findings indicate asymmetric junction at the molecular level in the present t(11;22). None of the reported conserved sequences that mediate other cancer chromosome translocations was observed around the genomic junctions. Instead, a palindromic hexamer 5'-GCTAGC-3' was found to flank the breakpoints of both genes on der(22), which may have a functional significance in the genesis of the t(11;22).