SummaryFollowing induction chemotherapy for acute myeloid leukaemia (AML), sensitive determination of minimal residual disease (MRD) in patients achieving complete remission (CR) should enable the detection of early relapse and allow intervention at a more favourable stage than at overt relapse. We have determined the expression levels of the Wilms' tumour gene (WT1) by real-time quantitative polymerase chain reaction (RQ-PCR) in peripheral blood and bone marrow in 133 newly diagnosed AML patients and compared them with those in healthy volunteers. At diagnosis, the WT1 level exceeded normal expression in 118 of 133 (89%) patients, and was high enough to allow for detection of a WT1 decrease of least 1000-fold in 98 of 133 (74%) patients following induction therapy. Concomitant monitoring of fusion transcripts (PML-RARa, AML1-ETO, MLL-MLL, CBFb-MYH11, or DEK-CAN) in 38 patients identified different relationships between WT1 and fusion transcript levels, the AML1-ETO group showing remarkably low levels of WT1 compared with fusion transcript. In 32 patients analysed longitudinally there was close concordance between relapse and increased WT1 levels. Parallel longitudinal monitoring of WT1 and fusion transcript showed close correlation in 18 of 18 patients. We conclude that WT1 expression by RQ-PCR may be employed as a tool to detect MRD in the majority of fusion transcript-negative AML patients.

The propensity of myelodysplastic syndrome (MDS) to transform into acute myeloid leukemia (AML) suggests the existence of common pathogenic components for these malignancies. Here, four genes implicated in the development of AML were examined for promoter CpG island hypermethylation in cells from 37 patients with different stages of MDS. Aberrant methylation was detected by polymerase chain reaction amplification of bisulfite-treated DNA followed by denaturing gradient gel electrophoresis. The highest rate of methylation was found for p15INK4B (51%), followed by HIC1 (32%), CDH1 (27%), and ER (19%). Concurrent hypermethylation of > or = 3 genes was more frequent in advanced compared with early-stage MDS (P < or = 0.05), and hypermethylation of p15INK4B was associated with leukemic transformation in early MDS (P < or = 0.05). The median overall survival was 17 months for cases showing hypermethylation of > or = 1 genes vs. 67 months for cases without hypermethylation (P = 0.002). Specifically, promoter hypermethylation identified a subgroup of early MDS with a particularly poor prognosis (median overall survival 20 months vs. 102 months; P = 0.004). In multivariate analysis including stage and thrombocyte count, hypermethylation of > or = 1 genes was an independent negative prognostic factor (P < 0.05). These data suggest that hypermethylation of p15INK4B, HIC1, CDH1, and ER contribute to the development and outcome of MDS.

The Polycomb group (PcG) of genes is important for differentiation and cell-cycle regulation and is aberrantly expressed in several cancers. To analyse the role of deregulated PcG genes in acute myeloid leukaemia (AML), we determined by RQ-PCR the expression of the PcG genes BMI-1, MEL18, SCML2, YY1 and EZH2, and the downstream PcG targets HOXA4, HOXA9 and MEIS1 in diagnostic bone marrow samples from 126 AML patients. There was a general overexpression of the genes in AML patients compared to 20 healthy donors, except of HOXA4 and MEL18, which both displayed a wide range of expression levels within the AML subgroups. Among the AML patients with normal karyotype, a low HOXA4 level was associated with a shorter overall survival (P = 0.005). In addition, expression levels of MEL18 and EZH2 were significantly (P < 0.025) higher in patients with complex karyotype and lower in CBF-mutated patients. The t(8;21) vs. inv(16) positive patients showed significantly different expression of SCML2, BMI-1, YY1, HOXA9 and MEIS1 (P < or = 0.01). Comparisons between the PcG and PcG-regulated genes and a number of clinical and molecular data revealed correlations to genes involved in DNA methylation (DNMT1, DNMT3B), apoptosis (BAX, CASPASE 3) and multidrug-resistance (MDR1, MRP ) (P < 0.01). In conclusion, our data suggest that the role of PcG and PcG-regulated genes in leukaemogenesis varies between, as well as within karyotypic subgroups.

SummaryThe upfront application of molecular methods for identifying the fusion transcripts arising from balanced translocations in haematopoietic malignancies has several advantages: sensitivity is independent of its frequency, i.e. rare ones are not missed, cytogenetically cryptic aberrations are identified and it provides a platform for minimal residual disease (MRD) detection. Employing a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay identifying 27 fusion transcripts we prospectively analysed blood and/or bone marrow samples from 390 patients referred for diagnosis and treatment for acute leukaemia and chronic myeloproliferative disorders (CMPD) from a geographically well-defined region in Denmark. A total of 233 patients were diagnosed with acute myeloid leukaemia (AML), 95 with acute lymphoblastic leukaemia (ALL) origin and 62 patients were recorded as CMPD. Twenty-three percent AML, 32% ALL and 55% CMPD patients exhibited chromosomal aberrations detected by the multiplex RT-PCR. Cytogenetically cryptic translocations were seen in 15% of the cases. Conversely, the cytogenetic analysis identified chromosomal aberrations other than translocations in 45% of AML cases and 63% of ALL cases. We conclude that, while the fraction of translocation positive leukaemia patients in an unselected cohort is lower than hitherto believed, a molecular approach to their diagnosis is worthwhile, partly for identifying cryptic and rare translocations, partly for monitoring MRD.

No abstract