J. Moravcova et al., SIMPLE COMPETITIVE 2-STEP RT-PCR ASSAY TO MONITOR MINIMAL RESIDUAL DISEASE IN CML PATIENTS AFTER BONE-MARROW TRANSPLANTATION, Leukemia, 12(8), 1998, pp. 1303-1312
Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR
) assessing the amount of transcripts of the BCR/ABL gene, the molecul
ar marker of chronic myeloid leukemia (CML), is the only method sensit
ive enough for monitoring of minimal residual disease (MRD) in CML pat
ients after bone marrow transplantation (BMT). In this study we presen
t a simple modification of competitive Q-RT-PCR using natural competit
ors from cell lines K562 and BV173, The competitors were used in the f
orm of unpurified RNA in cell lysates which ensured their high stabili
ty. Mixing competitors and samples before RNA extraction eliminated pr
oblems with quantification of cDNA or RNA entering the competitive rea
ction and with checking for the RNA quality and reverse transcription
(RT) efficiency. The bulk of the malignant clone was expressed as the
number of leukemic cells in 10(6) leukocytes when the overproduction o
f the BCR/ABL mRNA in the cell lines we used as competitors was taken
into account. It was found to be 82-fold and 14-fold in K562 and BV173
, respectively, in comparison with 100% Ph-positive CML standard. The
assay reliability was verified by comparison of results with the mathe
matical model of competitive PCR. The assay is highly reproducible and
sensitive (10(-5)). Its accuracy was proved to be excellent in a wide
range of malignant cell concentrations. The method is demonstrated on
three CML patients suffering from MRD after BMT. In conclusion, this
method fulfills all criteria of competitive Q-RT-PCR. Because of its s
implicity it is suitable for clinical laboratories and due to the high
stability of the lysates used it may serve in the standardization of
results between different laboratories.