Chromosomal fusion of the EWS gene to one member of the ETS family of transcription factors is the genetic hallmark of ES-. Detection of the chromosomal translocation by FISH, or amplification of the resulting fusion transcripts by RT-PCR, has become a well-established diagnostic component for molecular confirmation of histopathological tumor classification. Chromosomal breakage and re-fusion, however, occur in large intronic parts of the respective genes. Although the large size of the BCRs complicates the amplification of the breakpoint-spanning site, identification of the individual intronic DNA fusion sequence may unravel additional information associated with mechanisms involved in the rearrangement formation.
DNA-based tumor diagnostics has general advantages and disadvantages. The chemical stability of DNA facilitates storage and transport of patient material, and in contrast to RT-PCR methods, enables the detection of fusion genes independent of their gene expression. As a complementary diagnostic tool, DNA-based minimal residual disease detection could improve the quantification of resting residual tumor cells from different specimens including bone marrow, peripheral blood stem cell collection products, and paraffin-embedded, fixed-tissue sections. Fusion of EWS
to one of its partner genes is an early event in tumorigenesis and an essential oncogenic factor for maintaining the malignant transformation of ES- 
. In contrast to secondary genetic aberrations occurring during clonal evolution and contributing to the genetic heterogeneity within an individual ES- 
, the genomic fusion site remains a common and consistent molecular marker of tumor cells, unaffected by clonal selection from therapeutic intervention, and is currently evaluated as an additional tool for tumor cell quantification during treatment.
A major obstacle in quantifying DNA breakpoints in ES is the identification of the genomic EWS-FLI1
fusion sequence, because of large intronic regions within the BCRs. A classical approach, using multiple single PCRs to cover the complete BCRs, requires large amounts of patient material. This aspect and its consequent limit on the availability of adequate material for additional molecular diagnostics are probably the main reasons why only very few studies on genomic EWS-FLI1
fusion sequences are published, in contrast to numerous studies on EWS
transcripts and genomic breakpoints in fusion genes in diseases with more freely available material, e.g., acute leukemia 
. Two studies have identified the genomic fusion sequence in a total of eight individual cases of ES on the basis of transcript amplification, using primers specifically designed for the region of interest 
. A larger cohort of 77 Ewing- sarcoma patients was analyzed by Zucman-Rossi et al. using 15 single PCRs, but no patient characteristics are available and primer sequences have not been made permanently available 
In the present study, to overcome the problem of high DNA consumption, a nested MLR-PCR assay was established for reliable amplification of genomic EWS-FLI1
fusion sequences. Highly stringent primer selection and the use of advanced polymerases facilitated the development of a detection assay based on a single initial MLR-PCR. Thus, only minimal patient material is required to extract template DNA (100 ng), enabling the detection of tumor genomic fusion sequences from small tumor samples, e.g., fine needle biopsies. Using this assay EWS-FLI1
fusion sequences were identified from all 42 pediatric and young adult ES patients investigated. In addition, an analogous primer set was designed for detection of the second most common fusion gene in ES, the EWS-ERG
gene. The assay can be readily adapted to include rare fusion partner genes occurring in the remaining 2–5% of cases 
The patient-specific breakpoints are distributed in two subclusters within the EWS
-BCR, and this is in line with results from the cohort studied by Zucman-Rossi et al. 
. Despite searching for an extensive spectrum of sequence motifs and repeat elements, no DNA motif associated with either of the breakpoint clusters or with a significant subgroup of Ewing sarcoma was identified. However, small microhomologies and filler nucleotides at the fusion sites, as well as deletions or insertion of several nucleotides in the corresponding chromosomal derivatives (der22 and der11), resemble the characteristics of non-homologous end-joining (NHEJ) repair, and suggest that NHEJ repair is involved in translocation formation in ES 
A comparison of genomic breakpoint features of ES- with the far more extensively characterized breakpoints in leukemia and lymphoma reveals a number of similarities. In cells from hematological malignancy, different DNA double-strand break and repair mechanisms are associated with the lineage and differentiation stage of the cell population. Sequence-independent breakages as observed in ES- are also characteristic for immature lymphoid malignant cells 
. This subtype of leukemia cells shows small microhomologies or non-template insertions, as well as small deletions and duplications at the chromosomal breakpoint regions, indicating that NHEJ repair is involved in breakpoint initiation 
This observation of reciprocal balanced translocations with an NHEJ repair signature indicates molecular genetic similarities to mesenchymal tumors. It further supports results from functional and molecular genetic studies characterizing ES- by global gene expression profiling, which propose that ES- has its cellular origin in early mesenchymal progenitors 
In summary, in this study, genomic EWS-FLI1 fusion sequences were identified in a cohort of pediatric and young adult Ewing sarcoma patients with a specifically designed MLR-PCR assay requiring only minimal patient DNA. Detailed characterization of the genomic fusion sites revealed similarities to fusion site characteristics identified in immature lymphoid leukemia cells, and suggests that NHEJ repair mechanisms are involved in breakpoint initiation.