Genomic EWS-FLI1 Fusion Sequences in Ewing Sarcoma Resemble Breakpoint Characteristics of Immature Lymphoid Malignancies

Chromosomal translocations between the EWS gene and members of the ETS gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the EWS gene to FLI1, and is present in 85–90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic EWS-FLI1 fusion sites from as little as 100 ng template DNA. Characterization of the EWS-FLI1 fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5′ region of the EWS-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the FLI1-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma

Results for patients with sarcoma not otherwise specified and other diagnoses than Ewing sarcoma treated according to the Euro-EWING 99 trial

BACKGROUND: Euro-EWING 99 trial of the European Ewing tumor Working Initiative of National Groups (EE99) was an international phase III study in patients with Ewing sarcoma. The German Society of Pediatric Oncology and Hematology (GPOH) data center registered and followed patients with other diagnoses than Ewing sarcoma who were treated according to the EE99 protocol in an additional non-Ewing database. PROCEDURE: Data of 27 patients with other diagnoses than Ewing sarcoma treated according to the EE99 protocol were analyzed. Patients had miscellaneous histologic diagnoses, the majority were diagnosed with sarcoma not otherwise specified (NOS) arising in bone and soft tissue (63%). RESULTS: The median age at diagnosis was 16.9 years (range 4.5-41.4). Localized disease was diagnosed in 61.5% of the patients and 38.5% had distant metastases at time of primary diagnosis. The median follow-up time was 3.7 years (range 0.5-9.5). Patients with localized disease showed a 3-year event-free survival (EFS) of 68%, compared to 3-year EFS of 20% in patients with metastases (P = 0.042). Three-year EFS for patients with sarcoma NOS was 52%, patients with localized and metastatic disease showed 3-year EFS of 66 and 20%, respectively. CONCLUSION: EFS in patients with other diagnoses than Ewing sarcoma treated according to EE99 was significantly higher in patients with localized than metastatic disease. Sarcomas of soft tissue and bone that cannot be classified to current diagnostic categories constitute a therapeutic challenge

Genomic fusion site sequencing.

<p>(A) Genomic organization of the <i>EWS</i> and <i>FLI1</i> genes and corresponding breakpoint cluster regions (BCR). Nested primer sets for der22 are shown as double headed arrows. (B) Representative breakpoint sequencing workflow. Left: Gel electrophoresis of MLR-PCR products from two tumor samples in lane 1 and 2 (lane 3 negative control DNA; lane 4 ddH₂O; lane 5 positive control DNA; M = DNA ladder). Center: Gel electrophoresis of single long-range PCR products from 1^st round MLR-PCR product of sample 1 (lane 1–11; lane 12 positive control) to identify <i>FLI1</i> and <i>EWS</i> primers next to the fusion sites and to reduce amplification product size for direct sequencing. Right: Sequencing of the shortest amplification product and alignment to <i>EWS</i> and <i>FLI1</i> reference sequences.</p

The value of local treatment in patients with primary, disseminated, multifocal Ewing sarcoma (PDMES)

The value of local treatment in patients with primary, disseminated, multifocal Ewing sarcoma (PDMES) was investigated. We analyzed 120 patients registered into the European Ewing Tumor Working Initiative of National Groups (EURO-E.W.I.N.G. 99) trial at the trial center of Muenster from 1998 to 2006. Median age was 16.2 years. Local treatment of the primary tumor was surgery in 26 of 120 patients, surgery and radiotherapy in 21 patients, and definitive radiotherapy in 40 patients. For treatment of metastases, 6 of 120 patients received surgery; 9 patients, surgery and radiotherapy; and 33 patients, definitive radiotherapy. Forty-seven (39%) patients had local treatment of both the primary tumor and metastases, 41 (34%) patients of either the primary tumor or metastases, and 32 (27%) received no local therapy. Event-free survival (EFS) at 3 years was 0.24 (95% CI, 0.16-0.33). Univariate analyses demonstrated the impact of local therapy given to the primary tumor: 3-year EFS was 0.25 with surgery, 0.47 with surgery and radiotherapy, 0.23 with radiotherapy, and 0.13 when no local therapy was administered (P < .001). Three-year EFS in PDMES was also influenced by the local treatment: surgery, 0.33; surgery and radiotherapy, 0.56; radiotherapy, 0.35; no local therapy, 0.16 (P = .003). Three-year EFS was 0.39 in patients who received local treatment of both primary tumor and PDMES, compared with 0.17 in patients with any local treatment of either primary tumor or PDMES and 0.14 in patients with no local therapy (P < .001). Multivariate analysis showed absence of local treatment to be the major risk factor (HR = 2.21; P = .027; n = 20). Local therapy of involved sites is important for patients with PDMES and should complement systemic treatment whenever possibl

Breakpoint distribution in the BCR of <i>EWS</i> and <i>FLl1</i>.

<p>(A) Vertical bars above or below the breakpoint regions indicate individual breakpoint positions of Ewing sarcoma patients. Black boxes represent exons, and gray boxes correspond to repeat elements. (B) Results of Kernel density analysis (dashed line = breakpoint density; gray line = lower limit of 95% confidence band determined by bootstrapping procedure; black line = 95% confidence interval of a density function resulting from simulations at randomly distributed pseudo-breakpoints). X-axes indicate the BCR nucleotide positions within the respective reference gene. (C) Scatterblot of gender-specific <i>EWS-FLI1</i> breakpoints. Circles represent female, and squares represent male subjects. (D) Number of microhomologies and filler nucleotides at <i>EWS-FLI1</i> (der22; black bar) and <i>FLI1-EWS</i> (der11; gray bar) fusion sites. Each bar on the x-axis represents one individual.</p

Genomic <em>EWS</em>-<em>FLI1</em> Fusion Sequences in Ewing Sarcoma Resemble Breakpoint Characteristics of Immature Lymphoid Malignancies

<div><p>Chromosomal translocations between the <i>EWS</i> gene and members of the <i>ETS</i> gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the <i>EWS</i> gene to <i>FLI1</i>, and is present in 85–90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic <i>EWS-FLI1</i> fusion sites from as little as 100 ng template DNA. Characterization of the <i>EWS-FLI1</i> fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5′ region of the <i>EWS</i>-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the <i>FLI1</i>-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma.</p> </div

Patientś characteristics and breakpoint coordinates of tumor samples and cell lines.

<p>Patientś characteristics and breakpoint coordinates of tumor samples and cell lines.</p

Management and Outcome of Ewing Sarcoma of the Head and Neck

Ewing sarcoma (EWS) of the head and neck is rare. Multimodal treatment consists of chemotherapy and local treatment; however, local treatment for EWS of the head and neck is challenging. The first objective was to describe local treatment administered to the patients with localized EWS of the head and neck according to the EURO-E.W.I.N.G.99-trial, and to assess the impact on survival. The second objective was to systematically review the scientific literature available for this topic. Fifty-one patients were included. Local control consisted of surgery and/or radiotherapy (RT). Event-free survival (EFS) and overall survival (OS) were determined. Outcome was analyzed by comparing local treatment approaches. A Medline search was performed for EWS of the head and neck. Eighty-six percent of patients had localized disease. Most common primary sites included the skull (45%), maxilla (14%), and mandible (12%). Three-year EFS was 74% and 3-year OS was 87% for patients with localized disease. EFS was 40% for patients >15 years compared to 81% for patients <15 years. Local control consisted of surgery (S; 33%), RT (18%), or S + RT (45%). Related 3-year EFS was 81% (S), 80% (RT), and 72% (S + RT); 3-year OS was 80%, 76%, and 81%, respectively. In patients with EWS of the head and neck, age, and stage are important prognostic factors. Although not statistically significant, large tumor volume seems to be a negative prognostic factor. No difference in EFS and OS could be found when comparing patients treated with surgery, RT, or combined surgery and R