Objective: To evaluate the value of real time quantitative RT-PCR (Q-PCR) for monitoring bcr-abl mRNA levels in chronic myeloid leukemia (CML) patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).
Methods: Quantification of bcr-abl mRNA was performed on 316 bone marrow samples from 112 patients with CML after HSCT by Q-PCR using the TaqMan probe system. The bcr-abl mRNA level was normalized by control gene abl. Cytogenetic response was evaluated with fluorescent in-situ hybridization (FISH).
Results: The reproducible sensitivity of Q-PCR was 5 copies. The coefficients C(T) of interassay and intraassay variation for abl and bcr-abl were all below 2.0%. 289 bone marrow samples were collected from 101 CML patients who achieved a sustained complete cytogenetic response (CCyR) one month post allo-HSCT in a period of 6 - 60 months (median 12 months) at different intervals. In general, the median bcr-abl levels gradually decreased with the prolongation of time after HSCT: the median bcr-abl levels were 0.035% (0 - 0.406%) at 1 month post allo-HSCT (+ 1 month), 0.006% (0 - 0.683%) at +3 month, 0% (0 - 0.225%) at +6 month and remained 0% till +24 months. The highest level in CCyR patients detected at + 6 month was 0.068%. The bcr-abl mRNA level was decreased by 3 log in sustained CCyR patients at + 1 month compared with the newly diagnosed CML-CP patients (33.0%, data unpublished). On the contrary, Q-PCR results ranged from 0.12% to 13.45% in 8 cytogenetic non-responders or relapsed patients post allo-HSCT. Among them, 5 patients' samples were collected 1 - 2 months before cytogenetic relapse, the results were ranged from 0.09% to 3.42%. If 0.09% was assumed 0.09% as a threshold, 9 sustained CCyR patients (8.9%) were tested once higher than that within 6 month after HSCT but decreased to 0% eventually. 2 blast crisis patients achieved CCyR within 1.6 and 3 months after HSCT, but hematological relapse occurred after 1 and 1.5 months, and their bcr-abl mRNA levels increased dramatically from 0% and 0.14% to 46.9% and 75.9% respectively.
Conclusions: Q- PCR is a sensitive, precise and reliable technique, and can be used to monitor CML patients post allo-HSCT regularly. Patients in blast phase of CML should be monitored more frequently.