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Quantitation of cyclin D1 over-expression using competitive fluorescent reverse transcription polymerase chain reaction

A tool for the differential diagnosis of mantle cell lymphoma

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Abstract

Mantle cell lymphoma is characterized by the presence of the t(11;14)(q13;q32) translocation that causes over-expression of the BCL-1 gene and consequent overproduction of its gene product cyclin D1. We have developed a competitive fluorescent reverse transcription polymerase chain reaction assay for the detection and semiquantitation of cyclin D1 over-expression. Using this assay a definitive ratio of the expression of cyclin D1 to cyclins D2 and D3 can be determined, provided good quality RNA is available. A single upstream primer derived from a consensus sequence found in cyclins D1, D2, and D3 was labeled at the 5′ end using a fluorescent dye. Downstream primers specific to cyclins D1 and D2 were designed and used in conjunction with a previously published D3 specific primer. The fluorescently labeled PCR products were separated by electrophoresis using an ABI 377 DNA sequencer. Fluorescence emitted from each product was used to determine the ratio of expression of cyclin D1 to D2 and D3 by assigning a dosage quotient [D1/(D2+D3)]. The mean dosage quotient recorded from samples representing 29 non-MCL patients was 0.03 (SD ± 0.03), the maximum value being 0.11. Samples from eight patients with a diagnosis of MCL generated values greater than 2. Calculation of a dosage quotient using this competitive fluorescent reverse transcription polymerase chain reaction assay allows unequivocal identification of patients with over-expression of cyclin D1, providing a new tool for the differential diagnosis of MCL.

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Authors and Affiliations

  1. Molecular Genetics, Old Pathology Building, Royal Devon and Exeter NHS Healthcare Trust, Barrack Road, EX2 5DW, Exeter, Devon, England, UK

    C. L. Wickham & S. Ellard

  2. Department of Histopathology, York District Hospital, Wigginton Road, YO31 8HE, York, England

    H. Armitage

  3. Department of Haematology, Royal Devon and Exeter NHS Healthcare Trust, Barrack Road, EX2 5DW, Exeter, Devon, England

    M. V. Joyner & M. Boyce

  4. Department of Histopathology, Royal Devon and Exeter NHS Healthcare Trust, Barrack Road, EX2 5DW, Exeter, Devon, England

    P. Sarsfield

  5. Department of Pathology, School of Clinical and Laboratory Sciences, Royal Victoria Infirmary, NE1 4LP, Newcastle upon Tyne, England

    B. S. Wilkins & D. B. Jones

  6. Institute of Biomedical and Clinical Science, Peninsula Medical School, Barrack Road, EX2 5AX, Exeter, Devon, England

    S. Ellard

Authors
  1. C. L. Wickham
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  2. H. Armitage
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  3. M. V. Joyner
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  4. P. Sarsfield
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  5. M. Boyce
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  6. B. S. Wilkins
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  7. D. B. Jones
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  8. S. Ellard
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Correspondence to C. L. Wickham.

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Wickham, C.L., Armitage, H., Joyner, M.V. et al. Quantitation of cyclin D1 over-expression using competitive fluorescent reverse transcription polymerase chain reaction. Med Oncol 20, 77–85 (2003). https://doi.org/10.1385/MO:20:1:77

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  • DOI: https://doi.org/10.1385/MO:20:1:77

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