Research And Development Of Real-time Quantitative RT-PCR For Measurement Of Bcr-abl MRNA Transcripts

Postgraduate: Chen Jiansen Supervisor: Zhuo GuangshengOBJECTIVES: Chronic myeloid leukemia(CML) is a clonal neoplastic disorder of hematopoietic stem cells, which characterized, in most more than 95% of the cases, by t(9;22) (q34;q22) translocation, produces the bcr-abl fusion gene. The bcr-abl gene encodes a chimeric protein with elevated tyrosine kinase activity, which plays an important role in the pathogenesis of the disease. We developed and evaluated a rapid and reliable real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) approach using LightCycler technology for detection and quantification of bcr-abl transcripts in CML patients at diagnosis, during therapy and after bone marrow transplant(BMT).METHODS: The primers were designed to amplify the most frequent bcr-abl transcripts including b3a2, b2a2, e1a2 and some rare transcripts such as b3a3 and b2a3. A couple of LightCycler probes complementary to abl exon 3 was also designed, enabling detection and quantification of the bcr-abltranscripts and the normal abl transcript as an internal control. After optimized the conditions, sensitivity, specificity and reliability were evaluated. Standard curves were also plotted using the 10-fold dilution of recombined plasmid. To determine the utility of the assay, we quantified the bcr-abl/abl ratio in 45 peripheral blood (PB) samples (25 samples at chronic phase, 6 samples at accelerated phase and 14 samples at blast crisis) from 38 CML patients, a CML patient treated with bone marrow transplant (BMT) and another CMLpatient treated with Glivec(STI571) at accelerated phase were also monitored with RQ-PCR.RESULTS: By RQ-PCR, conditions were optimized to amplify less than 100 target molecules/reaction and detect one K562 cell in 105 cells from healthy donors. The intra-assay reproducibility of bcr-abl and abl transcripts resulted in coefficient variations of 0.68% and 0.59% of CT, and 12.93%, 8.6% of the respective concentrations. The inter-assay comparison were1.14% and 1.01% of CT, 18.89% and 12.72% of the respective concentration. The median value of bcr-abl and bcr-abl/abl ratio were (11542±5106) copies/ug total RNA and (10.58±5.12)% at chronic phase, (83350±7844) copies/ug total RNA and (84.20±3.78)% at accelerated phase, (79112±7956) copies/ug total RNA and (80.15±4.16)% at blast crisis. In a post-BMT patient and a patient treated with Glivec the bcr-abl/abl ratio were also correlated well with their clinic phases. CONCLUSIONS: We had,, established a real time fluorescent quantitative PCR and concluded that this RQ-PCR procedure is a reliable and sensitive method of monitoring CML patients during after therapy, and the bcr-abl/abl ratio correlates well with the clinic stages.