Quantitative competitive reverse transcriptase-polymerase chain reaction for BCR-ABL on Philadelphia-negative leukaphereses allows the selection of low-contaminated peripheral blood progenitor cells for autografting in chronic myelogenous leukemia

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-α) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients had 34% and seven patients >34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers ( r = 0.84, P < 0.0001) or bcr-abl/abl ratio ( r = 0.86, P < 0.0001) was found. for patients that underwent the procedure in early chronic phase, ph-negative collections showed different levels of bcr-abl expression. bcr-abl transcript numbers varied from a median of 100/μg rna in the first and second leukaphereses, to 500/μg rna in the third and fourth leukaphereses, and 1500/μg rna in the fifth leukapheresis ( P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (≤0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (χ 2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.