Abstract
The Ewing family of tumors (ET) generally contain translocations involving the EWS gene and the FLI or ERG genes. Identification of the translocation confirms the diagnosis of ET. Currently, diagnosis of the translocation is made by several methods. In general, these methods require different primer sets for amplifying different translocations and subsequent efforts to identify the amplified product. The need to employ different sets of primers to amplify different translocation junctions presents some limitations. We have developed a method based on PCR with consensus primers followed by direct automated sequencing of the amplified product. With this method we have correctly determined known as well as unknown ET-associated EWS-FLI and EWS-ERG translocations in appropriate specimens. Use of our consensus primers eliminates the need for separate PCRs to amplify EWS-FLI and EWS-ERG translocation junctions, and because direct sequencing is used for confirming the identity of the amplification product, the accuracy of detection becomes 100%. The method might also accurately diagnose ET-associated translocations other than EWS-FLI and EWS-ERG translocations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Unni, K. K. (1996) Dahlin’s Bone Tumors, Lippincott-Raven, Philadelphia, PA, pp. 249–261.
Delattre, O., Zucman, J., Plougastel, B., et al. (1994) Gene fusion with an ETS DNA-binding domain caused by chrosome translocation in human tumours. Nature 359, 162–165.
Sorensen, P. H. B., Lessnick, S. L., Lopez-Terrarda, D., Liu, X. F., Triche, T. J., and Denny, C. T. (1994) A second Ewing’s sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat. Genet. 6, 146–151.
Ishida, S., Yoshida, K., Kaneko, Y., et al. (1998) The genomic breakpoint and chimeric transcripts in the EWSR1-ETV4/E1AF gene fusion in Ewing sarcoma. Cyto. Cell Genet. 82, 278–283.
Peter, M., Couturier, J., Pacquement, H., et al. (1997) A new member of the ETS family fused to EWS in Ewing tumors. Oncogene 14, 1159–1164.
Urano, F., Umezawa, A., Hong, W., Kikuchi, H., and Hata, J. (1996) A novel chimera gene between EWS and E1A-F, encoding the adenoivirus E1A enhancer-binding protein, in extraosseous Ewing’ sarcoma. Biochem. Biophys. Res. Commun. 219, 608–612.
Adamas, V., Hany, M. A., Schmid, M., Hassam, S., Brinter, J., and Niggli, F. K. (1996) Detection of t(11;22)(q24; q12) translocation breakpoint in paraffin-embedded tissue of the Ewing’s sarcoma family by nested reverse transcription-polymerase chain reaction. Diagn. Mol. Path. 5, 107–113.
Barr, F. G., Xiong, Q. B., and Kelly, K. A. (1995) Consensus polymerase chain reaction, oligonucleotide hybridization approach for the detection of chromosomal translocations in pediatric bone and soft tissue sarcomas. Anat. Path. 104, 627–633.
Meier, V. S., Kuhne, T., Jundt, G., and Gudat, F. (1998) Molecular diagnosis of Ewing tumors, improved detection of EWS-FLI-1 and EWS-ERG chimeric transcripts and rapid determination of exon combinations. Diagn. Mol. Path. 7, 29–35.
Pfleiderer, C., Zoubek, A., Gruber, B., et al. (1995) Detection of tumour cells in peripheral blood and bone marrow from Ewing tumor patients by RT-PCR. Int. J. Cancer 64, 135–139.
Schlott, T., Nagel, H., Ruschenburg, I., Reimer, S., and Droese, M. (1997) Reverse transcriptase polymerase chain reaction for detecting Ewing’ sarcoma in archival fine needle aspiration biopsies. Acta Cytol. 41, 795–801.
Zoubek, A., Ladenstein, R., Windhager, R., et al. (1998) Predictive potential of testing for bone marrow involvement in Ewing tumor patients by RTPCR, A preliminary evaluation. Int. J. Cancer 79, 56–60.
Weber, K., Bolander, M. E., and Sarkar, G. (1998) PIG-B, a homemade monophasic cocktail for the extraction of RNA. Mol. Biotechnol. 9, 73–77.
Zucman, J., Melot, T., Desmaze, C., et al. (1993) Combinatorial generation of variable fusion proteins in the Ewing family of tumors. EMBO J. 12, 4481–4487.
Diffin, F., Porter, H., Mott, M. G., Berry, P. J., and Brown, K. W. (1994) Rapid and specific diagnosis of t(11;22) translocation in pediatric Ewing’s sarcoma and primitive neuroectodermal tumors using RNA-PCR. J. Clin. Path. 47, 562–564.
Bonin, G., Scamps, C., Turc-Carel, C., and Lipinski, M. (1993) Chimeric EWS-FLI1 transcript in a Ewing cell line with a complex t(11;22;14) translocation. Cancer Res. 53, 3655–3657.
Ida, K., Kobayashi, S., Taki, T., et al. (1995) EWS-FLI-1 and EWS-ERG chimeric mRNAs in Ewing’s sarcoma and primitive neuroectodermal tumors. Int. J. Cancer 63, 500–504.
Backer, A., Mount, S. L., Zaeka, M. A., et al. (1998) Desmoplastic small round cell tumor of unknown primary origin with lymph node and lung metastases, histological, cytological, ultrastructural, cytogenetic and molecular findings. Virchows Arch. 432, 135–141.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Morishita, T., Bolander, M.E., Zhang, K. et al. A method for accurate detection of translocation junctions in ewing family of tumors. Mol Biotechnol 18, 97–104 (2001). https://doi.org/10.1385/MB:18:2:097
Issue Date:
DOI: https://doi.org/10.1385/MB:18:2:097