Quantification of BCR-ABL transcripts by QC-RT-PCR in leukapheresis-collected blood progenitor cells in early diagnosed patients with CML

The quality of the graft is thought to be essential for a good outcome in aulologuus stem cell transplant for CML. We have recently demonstrated that a) Ph-negative peripheral blood progenitor cells (PBPC) can be collected by leukapheresis during early recovery after chemotherapy-induced marrow aplasia, b) these cells are able to restore nORnal polyclonal hemopoiesis after autografting and c) the best clinical results are achieved in patients autografted with Ph-negative PBPC In order to monitor minimal residual disease in Ph-negative PBPC, we set a quantitative-competitive RT-PCR assay for BCR-ABL and analyzed Ph-negative collections in 18 patients mobilized in the first year from diagnosis without previous IFN-a treatment. We generated a construct m which the fusion region of the BCR-ABL gene was cloned in pCR-II vector and a 222 bp of foreign DNA was inserted in the ABL portion of the cDNA. This construct was used as competitor DNA to estimate BCR-ABL and ABL transcripts in 147 samples Ratio between the t\u> BCR-ABL and ABL was calculated for every sample. Statistical correlation between cytogenetics and QC-RT-PCR was higher than 0.8. Ph-negative collection from the same patients showed more than one log of difference in the amount of BCR-ABL transcripts, with the lower levels in the first two teukaphereses Sample BCR-ABL trans/ugRNA (median) BCR-ABL/ABL diagnosis 50000 0.3 1st leuk. 100 0.002 2nd leuk. 100 0.002 3rd leuk. 500 0.006 4th leuk 500 0.01 Sthleuk 1500 0.01 Indeed, the BCR-ABL level found in the first leukaphereses of these patients was lower than in the corresponding samples from a group of patients pre-treated for more than 12 months with IFN-α therapy. Patients mob BCR-ABL Irans/BCR-ABL/ABL agRNA fmedl in the 1 st leuk. (med) £12 mo. fromdx.inoINF 100 0.002 >12mo.IFN 1000 0.01 In conclusion, quantitative-competitive RT-PCR for BCR-ABL has been proven to be a powerful tool in monitoring leukemic contamination in PBPC collections. Follow-up data of patients autografted with low-contamination PBPCs are necessary to establish the clinical relevance of this selection.