SPECIFIC AND SENSITIVE DETECTION OF THE EWS FLI1 FUSION PROTEIN IN EWINGS-SARCOMA BY WESTERN BLOTTING/

We applied Western blotting, using an antibody against the carboxy ter minal of the FLI1 protein, for detection of the 68-kDa EWS/FLI1 fusion protein in cultured Ewing's sarcoma cells and in four surgical biopsi es of Ewing's sarcoma. Of six different human cell lines, the 68-kDa f usion protein was identified only in Ewing's sarcoma cells carrying th e t(11;22)(q24;q12) translocation. The four samples from Ewing's sarco ma patients were also found to contain the 68-kDa fusion protein. The lowest detection level for total protein loaded on the gel was 0.3 mu g. When whole Ewing's sarcoma cells were used for Western blotting wit hout prior protein extraction, the lowest detection level was 1,300 ce lls. It will be possible to use Western blotting for detection of the EWS/FLI1 fusion protein in the diagnosis of Ewing's sarcoma in surgica l biopsy specimens, and possibly also in fine-needle aspirates. As the method is not dependent on the quality of mRNA in the sample and invo lves no risk of contamination, it will be a powerful complement to the reverse-transcription polymerase chain reaction (RT-PCR).