Advertisement

Molecular Biotechnology

, Volume 18, Issue 2, pp 97–104 | Cite as

A method for accurate detection of translocation junctions in ewing family of tumors

  • Toru Morishita
  • Mark E. Bolander
  • Kunbo Zhang
  • Susumu Tamai
  • Yoshio Mii
  • Gobinda Sarkar
Research

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.

Index Entries

Ewing’ tumor RT-PCR automated sequencing translocation consensus primer 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Unni, K. K. (1996) Dahlin’s Bone Tumors, Lippincott-Raven, Philadelphia, PA, pp. 249–261.Google Scholar
  2. 2.
    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.CrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    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.CrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    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.Google Scholar
  9. 9.
    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.CrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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.PubMedGoogle Scholar
  17. 17.
    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.PubMedCrossRefGoogle Scholar
  18. 18.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2001

Authors and Affiliations

  • Toru Morishita
    • 1
    • 2
  • Mark E. Bolander
    • 1
  • Kunbo Zhang
    • 1
  • Susumu Tamai
    • 1
    • 2
  • Yoshio Mii
    • 1
    • 3
  • Gobinda Sarkar
    • 1
  1. 1.Department of OrthopedicsMayo Clinic and Mayo FoundationRochester
  2. 2.Department of Orthopedic SurgeryNara Medical UniversityNara
  3. 3.Surgical CenterNara Medical University HospitalNara

Personalised recommendations