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Identification of PML/RARα rearrangements in suspected acute promyelocytic leukemia using fluorescence in situ hybridization of bone marrow smears: a comparison with cytogenetics and RT-PCR in MRC ATRA trial patients

Leukemia volume 14, pages 950–953 (2000)Cite this article

Acute promyelocytic leukemia (APL) is characterized by the reciprocal translocation t(15;17)(q22;q21) leading to the formation of PML/RARα and RARα/PML fusion genes.12 PML/RARα is believed to be critical for leukemogenesis23 but the role of RARα/PML transcripts, which are expressed in approximately 75% of cases4 remains to be determined. In patients with morphological APL the presence of a PML/RARα rearrangement predicts a favorable response to retinoids such as ATRA,5 which in combination with chemotherapy has been found to confer significant improvement in survival.6 Therefore establishing the presence of a PML/RARα rearrangement in suspected APL cases is valuable for optimal patient management, and for analysis of clinical trials. This is particularly important for the MRC ATRA trial, since all patients with a clinical diagnosis of APL were eligible for entry irrespective of the results of RT-PCR and cytogenetic analysis. Early studies in specialized centers suggested that the t(15;17) can be detected by cytogenetics in virtually all cases of morphological APL7 but large multi-center clinical trials, such as the MRC ATRA trial, have demonstrated that the t(15;17) is not detected by cytogenetics in at least 10% of cases in which PML/RARα rearrangements are demonstrated by molecular methods.48 Absence of the t(15;17) in these cases most commonly reflects poor quality metaphases of cryptic rearrangements due to insertion events documented by molecular techniques such as RT-PCR.3 In recent studies, fluorescence in situ hybridization (FISH) on metaphases and interphases from cytogenetic cultures has been shown to be a useful tool in identifying PML/RARα in APL patients at time of diagnosis and for minimal residual disease.910 In the present study we sought to determine whether bone marrow (BM) FISH, performed directly on BM smears, using PML and RARα probes could provide a more reliable indicator of the occurrence of PML/RARα rearrangements than conventional cytogenetics and we compared the efficacy of this approach with RT-PCR in a series of patients drawn from the MRC ATRA trial.

The present study considers 37 patients from the trial from whom BM smears were available for FISH analysis. Cytogenetic assessment was undertaken at local centers or at the central UK MRC AML trials cytogenetic service at University College Hospital, London, according to standard methods. In each case reported to have a normal karyotype, preparations were subject to at least 18 h of culture and a minimum of 20 metaphases were examined. Nested RT-PCR for the detection of PML/RARα and RARα/PML fusion transcripts was performed as previously described.11 PML/RARα FISH was performed as follows.

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Acknowledgements

SI, DG and molecular studies for the MRC ATRA trial were supported by the Medical Research Council and the Imperial Cancer Research Fund. The MRC AML trials tissue bank at University College Hospital, London was supported by the Kay Kendall Leukaemia Fund. We are also indebted to all the cytogeneticists involved in karyotyping these patients and to the clinicians who supplied material from patients entered into the MRC ATRA trial. Finally, we are grateful to Stephen Langabeer and Joanne Rogers for providing RNA for RT-PCR analyses.

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Authors and Affiliations

  1. Department of Haematology, Imperial College School of Medicine, London, UK

    S Iqbal, A Chase, JM Goldman & D Swirsky

  2. Department of Haematology, UCL Hospitals, London, UK

    D Grimwade & A Goldstone

  3. Department of Haematology, Division of Medical and Molecular Genetics, Guy's, King's and St Thomas’ School of Medicine, London, UK

    D Grimwade

  4. Department of Haematology, University of Wales, Cardiff, UK

    A Burnett

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  1. S Iqbal
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Iqbal, S., Grimwade, D., Chase, A. et al. Identification of PML/RARα rearrangements in suspected acute promyelocytic leukemia using fluorescence in situ hybridization of bone marrow smears: a comparison with cytogenetics and RT-PCR in MRC ATRA trial patients. Leukemia 14, 950–953 (2000). https://doi.org/10.1038/sj.leu.2401688

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