Use of Plasma RNA for Real-Time Quantitative RT-PCR to Monitor Imatinib Therapy in Patients with Chronic Myeloid Leukemia.
Academic Article
Overview
Research
Identity
Additional Document Info
Other
View All
Overview
abstract
AbstractMonitoring minimal residual disease (MRD) in chronic myeloid leukemia after imatinib therapy can help in determining therapeutic strategy. Sensitive methods based on the detection of the BCR/ABL fusion gene or its fusion transcript by FISH or RT-PCR, respectively, are used to monitor residual leukemia in peripheral blood or bone marrow cells. While sensitive, these methods require an invasive procedure to obtain bone marrow. The patchy nature of the residual disease may cause some of the reported variation in detecting MRD. We hypothesized that plasma RNA level reflects total body disease and is unaffected by the tendency of some myeloid cells not to circulate. We measured the BCR/ABL fusion transcript by real-time quantitative RT-PCR using RNA isolated from blood plasma. We used a quantitative RT-PCR assay capable of measuring both the b2a2 and b3a2 BCR/ABL fusion transcripts by targeting exon 13 of BCR with the 5 and exon 2 of ABL by the 3 primer. RNA isolated from 1030 l of frozen plasma was subjected to real-time quantitative RT-PCR using the Taqman technology. Quantification was achieved by extrapolation from a standard curve generated from a ten-fold dilution series of a synthetic oligonucleotide corresponding to the (+) strand of the target sequence. To normalize the fusion mRNA levels we also measured -actin mRNA from the same samples. Assays were run in duplicate. The results of plasma RT-PCR were correlated with results of conventional cytogenetic analysis, FISH, and quantitative RT-PCR of bone marrow suspension (bone marrow PCR). Plasma and bone marrow samples were obtained at 3, 6, 9, 12 and 15 months after imatinib therapy. Ninety-five plasma samples were analyzed. There was a strong positive correlation between the results of plasma RT-PCR vs. positive cytogenetics (n=95, p>0.00001), FISH (n=88, p>0.00001), and bone marrow RT-PCR (n=74, p>0.00001). At 12 months after imatinib therapy all but 2 of the 29 patients were in complete hematologic and cytogenetic remission (CR); one patient was in partial remission (PR) and one in hematologic, but not cytogenetic, remission. These two non-CR patients were positive by both bone marrow and plasma-based RT-PCR assay for the fusion transcript. Three of the four CR patients positive by both plasma and bone marrow PCR at 12 months progressed and converted to PR/minimal response at 15 months post-therapy. The level of plasma BCR/ABL fusion mRNA at 12 months was significantly higher (p=0.04) in these patients than in those who did not progress. Our results show that, by using 1030 l of plasma per assay, RT-PCR is at least as sensitive as cytogenetic or FISH analysis of bone marrow cells in monitoring MRD.
