SIMPLE COMPETITIVE 2-STEP RT-PCR ASSAY TO MONITOR MINIMAL RESIDUAL DISEASE IN CML PATIENTS AFTER BONE-MARROW TRANSPLANTATION

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.