Myeloid Leukemia

Real-Time RT-PCR Strategies 63
assess the value of MRD monitoring in a large number of patients enrolled in
clinical trials. Common guidelines and quality controls should be developed
within various international networks. This work is particularly essential for
communication between clinicians and molecular biologists. This standardization
should be obligatory for the assessment of prognostic value and for serial
assessment of MRD by RQ-PCR for rare FG, such as MLL-AF9, MLL-AF6, or
DEK-CAN. In these cases, centralization of results is recommended. This effort
should be started immediately for new markers such as WT1 or ras mutations.
In conclusion, it is evident that the technology is available for quantifying
gene transcripts within the clinical setting for the benefit of large series of
patients. In this way, it is possible to objectively document therapeutic efficacy
(MRD studies). This technology will definitively evolve, and here we have
described some variant approaches. However this technology is still in its
infancy, and is not yet implemented routinely in large multicenter trials for
myeloid malignancies. Two main limitations for its lack of penetration are
likely: first, improvements in standardization and better quality controls are
mandatory (such as the EAC program and the use of freeze-dried cells); second,
it must be available for the majority of the patients. Currently, fusion gene
transcripts are identifiable in only 35–45% of patients with AML. New markers
have to be found (new fusion transcripts, WT1, gene-expression profiles,
and so on). Very recently, Lossos et al. (49) showed that measurement of the
expression of six genes using RQ-PCR and based on gene-profiling data is
sufficient to predict overall survival in patients with large B-cell lymphoma.
All together, these efforts should pave the way towards the definition of new
biological markers—quantified gene transcript(s) for myeloid malignancies.
References
1. Laczika, K., Novak, M., Hilgarth, B., et al. (1998) Competitive CBFbeta/MYH11
reverse-transcriptase polymerase chain reaction for quantitative assessment of
minimal residual disease during postremission therapy in acute myeloid leukemia
with inversion(16): a pilot study. J. Clin. Oncol. 16, 1519–1525.
2. Tobal, K. and Liu Yin, J. A. (1998) Molecular monitoring of minimal residual
disease in acute myeloblastic leukemia with t(8;21) by RT-PCR. Leuk. Lymphoma
31, 115–120.
3. Barragan, E., Bolufer, P., Moreno, I., et al. (2001) Quantitative detection of
AML1-ETO rearrangement by real-time RT-PCR using fluorescently labeled
probes. Leuk. Lymphoma 42, 747–756.
4. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993) Kinetic PCR analysis:
real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026–1030.
5. Wittwer, C. T., Herrmann, M. G., Moss, A. A., and Rasmussen, R. P. (1997a)
Continuous fluorescence monitoring of rapid cycle DNA amplification.
Biotechniques 22, 130–131, 134–138.