How Best to Express the Results of Serial RQ-PCR Studies to Measure Minimal Residual Disease in Treated Leukaemia Patients?.

The use of real time quantitative PCR (RQ-PCR) is now established for monitoring minimal residual disease (MRD) in patients with leukemia responding well to therapy. The level of the target gene is usually expressed as a percentage ratio normalised to the control gene. Measurement of a control gene is needed to exclude the possibility of false negative values due to sample (RNA) deterioration. The ideal control gene should be expressed in blood and/or bone marrow at intermediate level without significant variation between different patients; its RNA should have stability similar to that of the gene of interest; and it should not have a processed pseudogene. For monitoring the low levels of BCR-ABL transcripts characteristic of Ph-positive CML during treatment, different laboratories have quantified as controls (amongst others) ABL, BCR, G6PD, GAPDH,β-actin and β₂-microglobulin. Despite the ABL gene being a component of the BCR-ABL fusion gene, it fulfils the criteria for a good control. Moreover as it is expressed at relatively low levels it is sensitive to sample deterioration. Analysis of 10,021 CML blood samples revealed that ABL transcript numbers were consistent when BCR-ABL transcript numbers were less than 20,000 (median 50,200), but above this level ABL quantification was influenced by BCR-ABL copy number (median 110,500). In MRD studies designed specifically to detect small numbers of residual transcripts, the contribution of amplifying BCR-ABL to the control value is not critical to the assessment of tumour load. A contemporaneous comparison of levels of BCR and ABL transcript levels in 831 blood samples taken from patients with CML at various stages of treatment showed a linear relationship (p=0.0001), but the median BCR expression was 0.7 log greater than the median ABL expression. In practice an assay using ABL as control gene for measuring the log reduction in BCR-ABL transcript levels following effective treatment compared with a BCR-ABL baseline value obtained before treatment yields values approximately equivalent to the log reduction obtained with an assay based on BCR as control gene. Nonetheless the variable expression of the commonly used control genes means that log reduction values may differ between centers. Thus we believe that the best method for reporting MRD values in CML patients undergoing treatment is as a percent ratio compared with previous values, i.e. the serial samples post treatment. Log reductions may be a useful ancillary for use within individual centres.