Fluorescent 5′‐exonuclease assay for the absolute quantification of Wilms' tumour gene (WT1) mRNA: implications for monitoring human leukaemias

Abstract

Summary. The Wilms' tumour gene (WT1) has been suggested as a powerful parameter for molecular monitoring of minimal residual disease (MRD) in leukaemias. However, molecular monitoring via WT1 RNA levels is far from being routinely performed, which is possibly owing to the complex and inaccurate quantitative reverse transcription polymerase chain reaction (RT-PCR) procedures. Using a newlydeveloped quantitative real time RT-PCR, we measured WT1 transcripts in peripheral blood leucocytes of patients with acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid leukaemia (CML). While healthy blood donors did not show measurable amounts of WT1 transcripts, WT1 RNA levels were detectable in all types of leukaemia. Furthermore, intraindividual WT1 transcript kinetics were exclusively dependent on disease progression, treatment and subsequent disease outcome. Using this approach, we could distinguish between treatment response and failure within the first days of therapeutic intervention. Moreover, gradually rising WT1 levels over a period of weeks and months paralleled long-term disease progression and appeared to be a prognostic indicator for subsequent clinical relapse. A linear correlation between quantities of WT1 and bcr/abl fusion transcripts could be seen in CML. We conclude that quantitative assessment of WT1 transcripts using real-time PCR is an appropriate method for molecular monitoring of AML, ALL and CML, and can be used independently for both short-and long-term monitoring of leukaemia patients.

Real-time PCR quantification of major Wilms’ tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences

“…These findings have been previously described and are in concordance with results of other studies. [47][48][49][50][51][52][53][54][55] WT1 isoform expression patterns showed at first glance subtle but significant differences among normal BM, AML and MDS, and also between childhood and adult samples. These specific patterns enabled the clustering of samples into groups representing the individual diagnoses.…”

Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study

“…[26][27][28] QRT-PCR primers and probes The fluorescent probe, 5 0 -6-FAM-CAACGCCCATCCTCTGCG GAGCCCA-TAMRA-3 0 (exon 6, nt position: 1230-1254, NM_ 024426), was used as described earlier by Kreuzer et al 29 The primers and probes were synthesized by TIB Molbiol, Berlin, Germany.…”

Poor correlation of kinetics between BCR-ABL and WT1 transcript levels after allogeneic stem cell transplantation

“…A total of 227 samples (185 PB and 42 BM) were analyzed, with a median of seven samples (range [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] per patient. PB samples from 13 healthy volunteers were used as controls.…”

“…However, in recent years, the use of Wilms' tumor gene (WT1) as a 'panleukemic' marker has been investigated in various studies. [7][8][9] WT1 encodes a transcription factor involved in the pathogenesis of Wilms' tumor. Further analyses of this gene have shown that the level of expression is high in most patients with acute leukemia and CML.…”