Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation.

Abstract

To analyze the value of real time RT-PCR for monitoring of bcr-abl expression in CML patients after allogeneic or autologous stem cell transplantation (SCT), we generated pairs of PCR-primers and TaqMan probes specific for either the b2a2- or the b3a2-variant of bcr-abl. Either variant could be detected specifically from cDNA from a single K562 (b3a2) and BV173 (b2a2) cell with the respective TaqMan probe. Bcr-abl expression was normalized by comparison with GAPDH expression, and samples were quantitated using standard cDNA dilutions from K562 or BV173 cells. In a retrospective analysis 13 patients with CML after allogeneic (n = 10) or autologous (n = 3) SCT including patients with relapsed or persistent CML were analyzed by both real-time and conventional nested RT-PCR. In addition chimerism was monitored by FISH analysis of sex chromosomes in three patients with relapsed disease. The bcr-abl/GAPDH ratio dropped at least 1000-fold in all seven patients evaluable prior to and after allogeneic SCT as estimated by real-time RT-PCR, and conventional RT-PCR became negative in 6/7 patients. In five patients with relapsed or persistent disease after allogeneic SCT the bcr-abl/GAPDH ratio eventually increased again, and real-time RT-PCR was as sensitive as conventional RT-PCR for detection of bcr-abl. Donor lymphocyte infusions (DLI) were given to all five patients, and the bcr-abl/GAPDH ratio dropped to undetectable levels in two patients both remaining in continuing molecular remission. In contrast, in three other patients the bcr-abl/GAPDH ratio decreased only or did not change significantly after DLI. In three patients undergoing autologous SCT the bcr-abl/GAPDH ratio dropped only 1.1 to 30-fold, and the patients were tested positive with real-time RT-PCR at all time points. These data demonstrate that real-time RT-PCR is valuable to quantitate bcr-abl expression in CML patients after transplantation.