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Abstract
Background
Following induction chemotherapy for AML, a sensitive determination of minimal residual disease (MRD) in patients achieving complete remission (CR) should enable the detection of early relapse. This study was designed to verify if quantitative assessment of the Wilms’ tumor (WT1) gene by real time polymerase chain reaction (RQ-PCR) can be used as a marker for MRD detection during the monitoring of AML.
Methods
WT1 gene expression was quantified by RQ-PCR in 31 patients with AML at diagnosis (27 patients) and during follow-up (29 patients) relative to ABL control gene. In four patients, the WT1 gene expression was analyzed in comparison to a second PCR marker, PML-RARA fusion transcript. Prognostic significance of WT1 gene expression was analyzed at diagnosis and at the primary CR evaluation. Longitudinal WT1 gene analysis was performed in 17 AML patients.
Results
At diagnosis, WT1 gene expression exceeded the control level in all of the patients. Higher levels of WT1 gene expression were not associated with shorter event free survival or overall survival at diagnosis. Higher levels of WT1 gene expression were associated with shorter event free survival after induction chemotherapy. Relapse was observed in eight of 17 patients analysed longitudinally, and an increase of WT1 gene expression preceded morphologic relapse in four patients with the fusion transcript negative. Concomitant monitoring of PML-RARA fusion transcript reveals the lack of a significant correlation with WT1 gene expression.
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Fig. 1.
WT1 amplication plot and standard curve. (A) Amplication plot of a 10-fold serial dilution of standard K562 cDNA (ranging from 10−5 to 100). The amplication plot shows an increase of reporter fluorescence during amplication. (x-axis, number of cycle; y-axis, relative fluorescence intensity). (B) Standard curve of K562 cDNA dilution for real-time PCR. The standard curve shows a linear correlation between the Ct value (y-axis) and the logarithm of the initial concentration of the standard K562 cDNA (x-axis).
Fig. 2.
WT1 expression at diagnosis (N=27) according to FAB subtypes.
Abbreviations: NC, Not classifiable by FAB classification.
Fig. 3.
WT1 expression at diagnosis (n=27) according to different molecular or cytogenetic prognostic subgroups of AML. Favorable: t(15;17), t(8;21), t(16;16); Intermediate: +8, +11, t(12;18) and normal karyotype; Adverse: −7, del(11)(q23), and complex karyotype as the presence of 3 or more abnormalities.
Fig. 4.
Kaplan-Meier plot of event free survival of patients with WT1 expression above or below 0.001.
Fig. 5.
Longitudinal monitoring of WT1 level in four exemplary patients with relapse. Arrows on the top of the graph indicate the time points of hematologic relapse.
Table 1.
Clinical characteristics in patients with acute myelogenous leukemia
Table 2.
Primers and probes
| Primers and probes∗ | Sequence; 5′ – 3′ |
|---|---|
| WT1 forward primer | GAT AAC CAC ACA ACG CCC ATC |
| WT1 reverse primer | CAC ACG TCG CAC ATC CTG AAT |
| WT1 probe | ACA CCG TGC GTG TGT ATT CTG TAT TGG |
| ABL forward primer | TGG AGA TAA CAC TCT AAG CAT AAC TAA AGG |
| ABL reverse primer | GAT GTA GTT GCT TGG GAC CCA |
| ABL probe | CCA TTT TTG GTT TGG GCT TCA CAC CAT T |
| bcr 3 forward primer | AGC TCT TGC ATC ACC CAG GGG A |
| bcr 1 forward primer | GTC TTC CTG CCC AAC AGC AAC C |
| common reverse primer | CTC ACA GGC GCT GAC CCC ATA GT |
| PML-RARA probe | CAG CCC TCC CTC GCC ACC CCC TCT A |
Table 3.
Prognostic significance of WT1 expression above or below 0.001 following induction chemotherapy (N=25)
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