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Quantitation of PML-RARα transcripts in APL patients

Leukemia volume 14, pages 1530–1531 (2000)Cite this article

B Cassinat et al1 reported the development of a real-time RT-PCR method for the quantitation of PML-RARα in APL patients with t(15;17). This paper raised a number of important points, which require the authors’ clarification to enable others to evaluate the technique reported.

  1. 1

    The authors reported the use of 5 μl of cDNA (equivalent to 0.25 μg of RNA) in their PCR. They also reported that 1 μg of RNA from NB4 cell line contains 104 copies of PML-RARα transcripts. This means that 0.25 μg of RNA (used in their PCR) of the undiluted NB4 cell line should contain 2500 copies of PML-RARα. If this is the case, then the best sensitivity they could achieve with their technique is one NB4 cell in 2.5 × 103 normal cells, and not the 10−6 or the 10−5 they reported to be able to detect consistently.

  2. 2

    Close examination of Figures 1 and 2 shows that the threshold cycles (CT) for 10−4 and 10−2 NB4 dilutions were 35 and 28 respectively which are equivalent to those shown for 102 and 104 PML-RARα plasmid copies. These data should therefore mean that 0.25 μg of RNA of 10−2 dilution of NB4 cells contains 104 copies of PML-RARα, and that 1 μg of RNA from undiluted NB4 cell line contains 4 × 106 copies of PML-RARα transcripts and not the 104 reported in the text. Did the authors make errors in their calculation of copy numbers or is there any other explanation?

  3. 3

    It would appear that the legends for Figures 1 and 2 were switched around.

  4. 4

    The authors continued to contradict their own data by suggesting in the text that they detected 104 PML-RARα copies in 1 μg of RNA from patient samples at diagnosis. Figure 3 shows that only one patient showed that level, while at least three other patients showed levels of approximately 105. Which is the correct level?

  5. 5

    The authors referred to a paper by Seale et al2 (Ref. 7) and suggested that this paper reported the detection of 103 PML-RARα copies in diagnosis samples of APL patients, which is not accurate. While Seale et al reported the detection of 103 copies of PML-RARα transcripts when using the inefficient short RT reaction, they also reported the detection of up to 9 × 106 PML-RARα copies in 1 μg of RNA when using the more efficient long RT reaction.

  6. 6

    In Figure 3 the level detected in the BM sample of the patient 4.5 months post diagnosis/5 months before relapse (800 copies) is equivalent to those detected in at least one other patient (patient 1 section a) 4 months post diagnosis, who went on to achieve complete remission. Patient 4, who also went on to achieve remission, showed a slightly lower level 4 months post diagnosis. Would the authors agree that the level of 800 copies shown by the patient in (b) was not predictive of relapse.

  7. 7

    The authors suggested that PML-RARα is poorly expressed, a suggestion put forward by a number of researchers when they could not achieve a sensitivity level above 10−4. Up to 9 × 106 copies/μg RNA is not a poor level of expression, and is equivalent to other genes such as AML1-MTG8.234 Furthermore, the ability of the authors and our group4 to achieve 10−6 sensitivity is evidence that PML-RARα is not poorly expressed as suggested.

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References

  1. Cassinat B, Zassadowski F, Balitrand N, Barbey C, Rain JD, Fenaux P, Degos L, Vidaud M, Chomienne C . Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR Leukemia 2000 14: 324–328

    Article  CAS  Google Scholar 

  2. Seale JRC, Varma S, Swirsky DM, Pandolfi PP, Goldman JM, Cross NCP . Quantitation of PML-RARα transcripts in acute promyelocytic leukaemia: explanation for the lack of sensitivity of RT-PCR for the detection of minimal residual disease and induction of the leukaemia-specific mRNA by alpha interferon Br J Haematol 1996 95: 95–101

    Article  CAS  Google Scholar 

  3. Tobal K, Newton J, Macheta M, Chang J, Morgenstern G, Evans PAS, Morgan G, Lucas GS, Liu Yin JA . Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse Blood 2000 95: 815–819

    CAS  Google Scholar 

  4. Tobal K, Macheta M, Liu Yin JA . Competitive RT-PCR for quantitating PML-RARα and monitoring minimal residual disease in acute promyelocytic leukaemia Haematologica 1999 84: (EHA4 Abstr. Suppl.) a418

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  1. University Department of Haematology, Molecular Oncology Group, Manchester Royal Infirmary, Oxford Road, Manchester, UK

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Tobal, K. Quantitation of PML-RARα transcripts in APL patients. Leukemia 14, 1530–1531 (2000). https://doi.org/10.1038/sj.leu.2401867

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