Detection of WT1 Expression in Bone Marrow Cells of Acute Leukemia Patients and during Their Follow-Up after Allo-BMT Using Real-Time Quantitative RT-PCR.

Abstract

WT1, a tumor suppressor gene originally found to be responsible for the development of childhood kidney tumor, is now thought to be also involved in the pathogenesis of human leukemia. Since WT1 is reported to be expressed at much higher level in bone marrow cells from MDS and all subtypes of leukemia comparing to the normal blood cells, it is proposed to be a “panleukemic marker” for the diagnosis of leukemia. The expression level of WT1 may also have significance in predicting prognosis and monitoring relapse. However, this notion on the usage of WT1 is somehow still remaining controversial because WT1 is also expressed to a certain level in a small population of CD34+cells. To measure more accurately the expression level of WT1 in bone marrow cells of acute leukemia patients (ALs). Real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and GAPDH expression levels in BM of 108 ALs and 23 non-leukemias patients by LightCycler. Besides, BM cells of 15 AL patients, including a total of 111 BM specimen taken during the follow-up after allo-BMT were also subject to RQ-RT-PCR. Normalized WT1 expression level (WT1N) was determined as a ratio between WT1 and GAPDH times 104. The expression levels of WT1N in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those of 23 ALs with complete remission(CR) and 23 non-leukemic controls (108.50±137.08 and 135.00±107.87 vs 3.88±3.09 and 2.23±3.19). Statistical differences were found neither between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, WT1N expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias, but there were no statistic differences among the M1, M2, M3 and ALL subtypes. Furthermore, the WT1N levels were not correlated to WBC counts in peripheral blood, the blast ratio in the BM and the expression of MDR-1 at presentation, but do correlated to the leukemia cell chromosome karyotypes. The dynamic changes of WT1N levels in 2 patients during treatment suggested that WT1 expression levels could be used to monitor the disease outcome and predict relapse. In gereral, the dynamic curves of WT1 level following allo-BMT were consistemt with the tendnecy of changes in expression levels of corresponding fusion genefor MRD monitoring. A re-increment of WT1 expression level during follow-up could be detected 40 to 180 days earlier to hematological relapse. These studies have provided strong evidence suggesting that WT1 expression levels in cells of AL patients were markedly higher than an undetectable or a marginal level expressed in non-leukemias patients. WT1 can indeed be a marker for evaluating therapy efficacy in leukemias. WT1 expression levels in leukemia patients following allo-BMT measured by real time RT-PCR can be a useful tool for monitoring MRD and warning the clinical relapse during follow-up.