Quantitative Assessment Of Wilms' Tumor Gene (WT1) MRNA By Real Time Quantitative PCR In Patients With Acute Leukemia And Chronic Myelogenous Leukemia

Postgraduate: Shourong Lin Supervision Guangsheng ZhuoObjectives To establish a method of fluorescent quantification polymerase chain reaction (FQ-PCR) for detecting WT1 mRNA. The measurement of WT1 mRNA by real time quantitative PCR in patients with acute leukemia (AL) and chronic myelogenous leukemia (CML) is to probe into the relationship between WT1 expression level and clinical phase of AL and CML. We performed an analysis of the WT1 transcript in patients with chemotherapy or Allo-BMT at sequential time intervals during follow-up and want to know whether the measurement of WT1 mRNA by FQ-PCR is a reliable method for monitoring minimal residual disease.Methods A real time PCR assay was developed to quantify WT1 mRNA in Periphal blood leukocytes or Bone marrow cells. We used a plasmid containing WT1 target sequence as an external standard. Tenfold serial dilutions of this plasmid yielded standard curve that allowed the quantification of WT1 gene copies. Bone marrow or peripheral blood derived cells of 47 AL patients, 39 CML patients and 10 healthy blood donors were studied for WT1 mRNA expression. ffT/-specific fluorescent quantification polymerase chain reaction (FQ-PCR) was performed.Results 1 .The standard curve indicated the linear relationship between CT (cycle threshold) and logarithm of template initial concentration, Y=-3.831X+43.54, r=-1.00. The within run variance and between run variance of low and high copies were 8.96%, 13.2% and 9.54%, 18.4%, respectively. The PCR method is sensitive to 1 : 104.2 WT1 mRNA was detectable in 35 of 45 (77.8%) newly diagnosed de novo AL patients. WT1 mRNA expressed in 24 of 27 (88.9%) newly diagnosed de novo AML patients and 11 of 18 (61.1%) newly diagnosed de novo ALL. WT1 mRNA wasundetected in 2 AL patients in complete remission. In 39 CML patients, WT1 mRNA was detectable in 8 of 21 (38.1%) Chronic Phase and 17 of 17 (100%) Acceleration Phase and Blast Crisis, WT1 mRNA was undetected in 1 post-Allo-BMT patient.Conclusions 1.The fluorescent quantification polymerase chain reaction for detecting WT1 mRNA has a good behavior of specificity, sensitivity and reproducibility. It is considered as a promising tool for MRD monitoring in leukemias lacking clone-specific markers.2.WT1 mRNA was expressed in the vast majority of patients with AL.The positive percentage of WT1 mRNA was 77.8%.3.The WT1 expression level increased in parallel with the progression of clinical phase in CML. The WT1 expression in chronic phase was low and the levels increased as the clinical phase progressed. The high level implied blast crisis.