Prognostic Value of Real-Time PCR BCR-ABL Transcript Monitoring after Allogeneic Stem Cell Transplantation in Chronic Myeloid Leukemia Patients.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) in chronic phase of chronic myeloid leukemia (CML) is associated with long-term disease-free survival and potentially eradication of leukemic cells. The goal of early MRD detection is to allow timely therapeutic intervention before hematologic relapse. The concomitant detection of BCR-ABL mRNA following alloHSCT is strongly associated with relapse, though not absolutely predictive. The relapse risk decreases with increased time after alloHSCT. The detection of BCR-ABL mRNA is the most strongly associated with relapse shortly after HSCT but all patients need to be monitored indefinitely after transplantation by molecular techniques presumably at 3–6 months intervals. Real-time quantitative PCR (RQ-PCR) for BCR-ABL mRNA provides an accurate and reliable measure of response to therapy in CML. In this study we evaluated 412 available samples from 75 patients at 1 month to 10 years after allo HSCT. Quantification of BCR-ABL was performed by RQ-PCR assay according to the Europe Against Cancer (EAC) protocol. Peripheral blood/bone marrow samples were studied every 3–6 months after alloHSCT for the presence of BCR-ABL transcripts using RT-PCR/nested PCR and RQ-PCR. RT-PCR positive patients were analyzed further at monthly intervals. RNA isolation from mononuclear cells was performed by column method. Reverse transcription was performed using Super Script II and random hexamers. BCR-ABL level was normalized with control ABL gene and expressed as the ratio of BCR-ABL/ABL compared to diagnostic sample or median expression values of BCR-ABL/ABL from EAC protocol. In our group BCR-ABL/ABL ratio decreased at least 1000-fold in all patients after alloHSCT. RT-PCR became negative in 64.7% patients after first 90 days. In the group of 65 patients with RQ-PCR tests performed at least 1 year after alloHSCT, 12 (18.5%) patients were always negative (no BCR-ABL/ABL transcripts detected, at least 10⁻⁵ test sensitivity), 40 (61.5%) were persistently low-level positive (with the BCR-ABL/ABL ratio less than 0.02%) and 13 (20.0%) patients were stable, high-level positive with transcript levels exceeded 0.02% threshold. The molecular relapse was observed in three patients with 15–80 fold increased of BCR-ABL expression in the first year after SCT. Decreased immunosuppressive therapy allowed achieving molecular remission in two patients. One patient developed hematological relapse despite donor lymphocyte infusion (DLI). One patient developed cytogenetic relapse and was successfully treated with 400 mg imatinib daily dose and achieved molecular remission with a low-level BCR-ABL expression. Standard imatinib therapy was applied in seven patients prior to SCT without negative transplant outcome. We conclude that RQ-PCR is valuable method to quantitate BCR-ABL expression in CML patients after alloHSCT and allows monitoring the kinetics of BCR-ABL mRNA transcipts and is useful in prediction of the hematologic relapse.