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The Ewing family of tumors (EFT) is an important group of pediatric malignancies with a guarded prognosis. Little is known about the heterogeneity of EFT cells, and the cellular origin of EFT is disputed. We now add evidence that EFT are heterogeneous by showing that EFT cells from spheres growing in serum-free medium are markedly more tumorigenic than adherently growing EFT cells. Furthermore, EFT cells strongly expressing CD57 (HNK-1), a surface marker for migrating and proliferating neural crest cells, are more tumorigenic than cells with low expression of CD57, possibly mediated in part by enhanced adhesion and invasion. We contribute to the controversy about the cellular origin of EFT by clonal analysis, showing that EFT cells can differentiate similar to neural crest cells. These data increase our knowledge about the pathogenesis and heterogeneity of EFT.

Giant-cell tumor of bone (GCT) is a locally aggressive neoplasm of unknown etiology and pathogenesis. Cytogenetically, no consistent chromosomal alterations, apart from telomeric associations involving various chromosome ends, have been described. Recently, however, it was reported that by using highly sensitive nested RT-PCR, a high proportion of GCT displays chimeric EWS/FLI1 fusion transcripts, i.e., the molecular genetic feature previously known to be strongly associated with the Ewing family of tumors. Thus, we decided to perform single-step and nested RT-PCR analyses on fresh frozen samples from 10 cases of GCT, all of which had also been subjected to cytogenetic analysis. After short-term culturing, none of the samples displayed any t(11;22)(q24;q12), the translocation characteristically giving rise to the EWS/FLI1 fusion, nor any other type of rearrangement of 11q24 or 22q12. Furthermore, in none of the cases did the RT-PCR analysis, whether single step or nested, result in products corresponding to a hybrid EWS/FLI1 transcript. On the basis of these results, we conclude that translocations leading to fusion of the EWS and FLI1 genes are not part of the pathogenesis of GCT.

Thde t( 11;22)(q24;q 12) and t(21;22)(q22;q 12) are specific chromosomal translocations found in the Ewinhd family of tumors including ES, PNET and Askin tumors. In these translocations, the amino-terminal portion of the EWS gene located in 22q 12 fuses to the carboxyl-terminal portion of the FLI-I gene located in 11 q24 or the ERG gene located in 21 q22, which belong to the ets oncogene super-family of transcription activators. We investigated the chimeric mRNAs of 15 ESs (7 cell lines and 8 tumor samples) and 7 PNETs (3 cell lines and 4 tumor samples) using the RT-PCR method and sequencing. We detected 2 types of EWS-ERG chimeric mRNA in 2 ES cell lines and I PNET tumor sample in addition to 4 types of EWS-FU-1 chimeric mRNA in 11 ESs (4 cell lines and 7 tumor samples) and 4 PNETs (2 cell lines and 2 tumor samples). There seemed to be no association between the type of chimeric mRNA and clinical features such as sex, age, primary site and histopathology of the patients. All of the chimeric mRNAs are generated from in-frame junctions and are thought to encode fusion proteins that may be the molecular mechanism involved in the Ewing family of tumors.

The Ewing family of tumors is recurrently characterized at the molecular level by the presence of a fusion transcript between the EWS gene on chromosome 22 and either the FL-I or ERG genes, 2 closely related members of the Ets family of transcription factors. We have investigated 12 primary human tumors, II xenografts and II cell lines, which have been shown to express chimeric EWS transcripts in search of p53 mutations. Fragments of exons 5 to 8 and the corresponding consensus splice sequences were amplified by PCR and analyzed by denaturing gradient gel electrophoresis (DGGE). In 12 of 34 samples p53 mutations were detected (including 4 samples with multiple p53 mutations). The distribution of the mutations in the various samples was as follows: primary tumors 2/12; cell lines 5/11; xenografts 5/11. No correlation between the presence or absence of p53 mutations and the presence of a specific EWS chimeric transcript was observed. In addition, we observed that p53 mutations were almost always associated with a second hit (either deletion or second mutation) on the other p53 allele. © 1994 Wiley-Liss, Inc.