Abstract. To date, approximately one half of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The Wilm's tumour gene (WT1) has been suggested as a possible molecular marker of MRD in AML. The expression of WT1 in peripheral blood (PB) was measured using quantitative real-time reverse transcription-polymerase chain reaction in peripheral leukocytes from 151 patients with AML at diagnosis. WT1 expression was significantly elevated, i.e. up to 3 orders of magnitude in the majority (80%) of AML patients at diagnosis compared to the PB of healthy donors. Sequence samples of the long-term followed-up AML patients treated with chemotherapy and/or allogeneic bone marrow transplantation were analysed for WT1 expression. The results revealed that the hematological relapses were preceded (median, 1.8 months) by an increase in WT1 gene expression. For the practical utility of this gene as a molecular marker of relapse, it was necessary to determine an upper remission limit, crossing which would signal hematological relapse. The upper remission limit was determined in our set of patients to be 0.02 WT1/ABL. The AML patients who consequently relapsed crossed this upper remission limit; however, those in permanent remission did not. Therefore, this upper remission limit could be taken as the border of molecular relapse of AML patients. Moreover, insufficient decline of WT1 expression under the upper remission limit following induction and/ or consolidation therapy was associated with markedly high risk of relapse. The results show that our upper remission limit can be taken as the border of molecular relapse of AML patients and WT1 levels following initial therapy as a beneficial prognostic marker.

Cyclins are very important components of the cell cycle machinery because their levels regulate cell proliferation. They have also been found to be prognostic factors in various cancers. We studied the expression of the positive cell cycle regulators (D cyclins) and the cell proliferation marker (Ki-67) in human acute myeloid (AML), chronic myeloid (CML), acute lymphoblastic (ALL) and chronic lymphocytic (CLL) leukemia [mainly by comparative reverse transcription polymerase chain reaction (RT-PCR)]. Both leukemic and normal cells were positive for cyclin D3 expression. Significant differences were found in the expression of cyclin D1, which was the highest in leukocytes (CD19 + ) of CLL patients whereas lower expression was found in CML, AML and ALL patients and normal bone marrow and peripheral blood leukocytes (P < 0.001). The higher expression of cyclin D1 in leukocytes of CLL patients compared to CML patients was confirmed by quantitative real-time RT-PCR with a TaqMan probe in a subset of CLL and CML patients. Differences in cyclin D1 expression between CLL and CML patients were also confirmed on protein levels by western blotting. Expression of the proliferative marker Ki-67 was high in CML, ALL and AML cells and low in CD19-positive CLL cells. The results demonstrate that the level of cyclin D1 negatively correlates with the proliferation properties of leukemic cells. We did not find any significant relationship between cyclin D1 expression in cells of CML and AML patients and their clinical outcome.

No abstract

Relapse remains the major cause of treatment failure in AML; however, RQ-PCR assays to detect leukemic fusion transcripts have been shown to identify reliably those patients at highest risk of relapse, allowing development of a more individualized treatment approach. In cases lacking a leukemia-specific MRD marker, quantification of genes over-expressed in AML e.g. Wilms’ Tumor gene (WT1) could provide more a precise measurement of disease response and quality of remission, potentially enhancing risk scores such as the one developed by the MRC used to identify those patients most and least likely to benefit from allogeneic transplant in first CR. WT1 is over-expressed in at least 75% of AML cases. Within the European LeukemiaNet we systematically evaluated 9 published and “in house” WT1 assays. Assays were excluded due to demonstrated lack of RNA-specificity or location within the 3′ region of the gene which has been shown to be subject to deletion or mutation in AML. An assay located within the 5′ region associated with superior sensitivity was ultimately selected following parallel testing in 11 labs and evaluated in 238 diagnostic peripheral blood (PB) and 386 bone marrow (BM) samples. WT1 was over-expressed in the majority, with comparable levels in PB and BM (PB - median 4637 WT1 copies/104ABL copies, range 0–1132709; BM - median 7212, 0–750571), as compared to normal BM (median 19.8, 0–213), PB (median 0.01, 0.01–47.6) and PBSCs (median 6.1, 0–39). In cases over-expressing WT1, kinetics of transcript reduction were evaluated following induction. A greater response was associated with a significantly reduced risk of relapse (hazard ratio 0.65 per log reduction (95% CI 0.43–0.96), p=0.03), although this failed to remain significant when adjusted for age, presenting WBC and cytogenetic risk group, which are key variables in the MRC risk index. Indeed, there was a highly significant correlation between larger log reduction in normalized WT1 transcript level and better risk score (p=0.0001). Sequential analysis of PB and BM samples from 15 AML cases with low WT1 expression (<250 copies) showed no significant modulation in transcript level on regeneration after chemotherapy, indicating that in WT1+ AML, transcript levels detected in follow-up samples reliably reflect disease status. This study provides evidence that recognized pre-treatment risk factors for relapse correlate closely with kinetics of response to induction therapy and lend support to the evaluation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification and identify those patients most suited to proceed rapidly to allogeneic transplant.