Myeloid Leukemia

Real-Time RT-PCR Strategies 61
Fig. 10. Decision algorithm for the interpretation of real-time quantitative polymerase
chain reaction results according to Europe Against Cancer criteria. For minimal
residual disease follow-up monitoring, the interpretation of the results depends on
the level of control gene expression for assessment of the quality of the sample. CG,
control gene; FG, fusion gene; CN, copy number; NA, not amplifiable; NQP, not quantifiable
positive; NEG, negative; POS, positive.
Thus, the interpretation of results varies depending on whether the sample
was obtained at diagnosis or during serial assessments for MRD. In all cases,
to ensure appropriate interpretation, it is essential that the RQ-PCR data that
have been obtained be assessed within the clinical context of the AML patients.
As an example, at diagnosis a low positivity result should always be suspected
of being false data.
5. Conclusion and Future Steps
RQ-PCR is the method of choice for quantifying mRNA levels of FG and
CG. This technology is fast, accurate, and sensitive compared to the other methods
of nucleic acid detection. However, RQ-PCR necessitates optimization and
control of the parameters relevant to the pre-analytic and analytical phases.
Until now, all RQ-PCR results to detect FG and CG in AML were obtained by
using dual labeled probe technology with relatively identical probes and primers
and with an identical instrument (ABI 7700). This quantitative method is
robust and accurate. However, the new chemistries and new instruments facilitate
multiplexing that combines the quantification of control gene and fusion gene in a
single PCR. Furthermore, they may give the same results with a lower cost.
In 2005, real-time PCR has become a technology that is essential in MRD
studies in patients treated for AML. Cumulative data suggest that quantitative