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An urgent need for new treatment modalities is emerging in elderly patients with acute myeloid leukemia (AML). We hypothesized that targeting VEGF might furnish an effective treatment modality in this population. Elderly patients with AML were randomly assigned in this phase 2 study (n = 171) to receive standard chemotherapy (3 + 7) with or without bevacizumab at a dose of 10 mg/kg intravenously at days 1 and 15. In the second cycle, patients received cytarabine 1000 mg/m(2) twice daily on days 1-6 with or without bevacizumab. The complete remission rates in the 2 arms were not different (65%). Event-free survival at 12 months was 33% for the standard arm versus 30% for the bevacizumab arm; at 24 months, it was 22% and 16%, respectively (P = .42). The frequencies of severe adverse events (SAEs) were higher in the bevacizumab arm (n = 63) compared with the control arm (n = 28; P = .043), but the percentages of death or life-threatening SAEs were lower in the bevacizumab arm (60% vs 75% of SAEs). The results of the present study show that the addition of bevacizumab to standard chemotherapy does not improve the therapeutic outcome of older AML patients. This trial is registered as number NTR904 in The Nederlands Trial Register (www.trialregister.nl).

Purpose: The treatment of acute myeloid leukemia (AML) in first relapse is associated with unsatisfactory rates of complete responses that usually are short lived. Therefore, a clinically useful prognostic index can facilitate therapeutic decision making and evaluation of investigational treatment strategies at relapse of AML., Patients and Methods: A prognostic score is presented based on the multivariate analysis of 667 AML patients in first relapse among 1,540 newly diagnosed non-M3 AML patients (age 15 to 60 years) entered onto three successive Dutch-Belgian Hemato-Oncology Cooperative Group and the Swiss Group for Clinical Cancer Research Collaborative Group trials., Results: Four clinically relevant parameters are included in this index (ie, length of relapse-free interval after first complete remission, cytogenetics at diagnosis, age at relapse, and whether previous stem-cell transplantation was performed). Using this stratification system, three risk groups were defined: a favorable prognostic group A (overall survival [OS] of 70% at 1 year and 46% at 5 years), an intermediate-risk group B (OS of 49% at 1 year and 18% at 5 years), and a poor-risk group C (OS of 16% at 1 year and 4% at 5 years)., Conclusion: The prognostic index estimates the outcome of AML patients in first relapse using four commonly applied clinical parameters and might identify patients who are candidates for salvage and investigational therapy.

Complete remission (CR) in patients with acute myeloid leukaemia (AML) is the primary endpoint for the evaluation of induction treatment and treatment strategies. However, the choice and application of the criteria for a haematological CR can often become a subject of debate because of regeneration more than 5% blasts may be present at the time of response evaluation; platelet and neutrophil recovery may be incomplete and marrow cellularity can vary. This study examined the individual parameters for CR in 1250 adult patients with de novo AML treated according to three successive study protocols. Patients with < or =5% blasts showed the best overall survival (OS) and the lowest relapse risk (RR). This was independent of blast cells present in the peripheral blood or bone marrow (BM) cellularity. In the same patient group, the presence of extramedullary leukaemia, incomplete platelet (<100 x 10(9)/l) or neutrophil (<1.0 x 10(9)/l) recovery caused a reduced OS and increased RR. In conclusion, < or =5% blasts in the BM, recovery of neutrophils and platelets, and the absence of extramedullary disease constitute the cornerstones for the definition of a haematological CR in patients with AML.

The proto-oncogene EVI1 encodes a DNA binding protein and is located on chromosome 3q26. The gene is aberrantly expressed in acute myeloid leukemia (AML) patients carrying 3q26 abnormalities. Two mRNAs are transcribed from this locus: EVI1 and a fusion of EVI1 with MDS1 (MDS1-EVI1), a gene located 5' of EVI1. The purpose of this study was to investigate which of the 2 gene products is involved in transformation in human AML. To discriminate between EVI1 and MDS1-EVI1 transcripts, distinct real-time quantitative polymerase chain reaction (PCR) assays were developed. Patients with 3q26 abnormalities often showed high EVI1 and MDS1-EVI1 expression. In a cohort of 319 AML patients, 4 subgroups could be distinguished: EVI1(+) and MDS1-EVI1(-) (6 patients; group I), EVI1(+) and MDS1-EVI1(+) (26 patients; group II), EVI1(-) and MDS1-EVI1(+) (12 patients; group III), and EVI1(-) and MDS1-EVI1(-) (275 patients; group IV). The only 4 patients with a 3q26 aberration belonged to groups I and II. Interestingly, high EVI1 and not MDS1-EVI1 expression was associated with unfavorable karyotypes (eg, -7/7q-) or complex karyotypes. Moreover, a significant correlation was observed between EVI1 expression and 11q23 aberrations (mixed lineage leukemia [MLL] gene involvement). Patients from groups I and II had significantly shorter overall and event-free survival than patients in groups III and IV. Our data demonstrate that high EVI1 expression is an independent poor prognostic marker within the intermediate- risk karyotypic group.

We have designed a real-time CBFB-MYH11 reverse transcription polymerase chain reaction (RT-PCR) assay to quantify minimal residual disease (MRD) in patients with inv(16)-positive acute myeloid leukaemia (AML). Six patients were followed for a median of 17.5 months after diagnosis during which 120 evaluable samples were analysed. The CBFB-MYH11 expression at diagnosis varied only fourfold between the six patients and was virtually identical to that observed in the CBFB-MYH11-positive cell line ME-1. For two cases, a patient-specific real-time PCR for CBFB-MYH11 quantification at genomic DNA level was designed. Similar disease levels were found at the RNA and genomic DNA level during and after treatment, indicating that CBFB-MYH11 gene expression was unaltered during treatment and that the percentage of malignant cells can be accurately quantified at the RNA level. Following successive courses of chemotherapy, the reduction of malignant cells was found to be significantly more pronounced (80-250-fold greater) in peripheral blood compared with bone marrow in five out of six cases tested. Treatment with gemtuzumab ozogamicin as sole agent at relapse did not result in a selective decrease of tumour cells in three cases analysed. We conclude that real-time PCR is a powerful method of monitoring MRD levels and quantifying the antileukaemic effect of separate (experimental) courses of chemotherapy.

The translocation (8;21) is a chromosome abnormality associated with acute myeloid leukemia (AML). As a consequence of the translocation the AML1 (CBFA2) gene in the 21q22 region is fused to the ETO(CDR,MTG8) gene in the 8q22 region, resulting in one transcriptionally active gene on the 8q- derivative chromosome. In this report we demonstrate the use of a highly specific dual-colour FISH method for the detection of t(8;21) on interphase cells. Genomic probes able to detect the chimeric AML1/ETO gene on the 8q- derivative chromosome were assayed on both normal and leukemic bone marrow and peripheral blood samples. Cut-off values were established by independent analysis of 15 bone marrow specimens negative for the translocation. The cut-off value of positive nuclei was determined to be 2% and the cut-off value for both positive nuclei and nuclei of uncertain classification, 4%. Persistence of cells above these cut-off values was interpreted as persistence of the mutated clone. A total of 36 samples at different disease stages were tested. Interphase cytogenetics detected the translocation at the onset and relapse in the BM or the PB of 14 AML patients with t(8;21). The technique appears to be an alternative tool to both conventional cytogenetics and reverse transcription polymerase chain reaction (RT-PCR) for the monitoring of disease during patients' follow-up. By enabling the analysis of individual cells, interphase FISH is ideal for clonality studies both for clinical and experimental applications.

With the use of molecular techniques it is now possible to define even subtle chromosomal abnormalities and the fusion products resulting from translocations. Defined clinical correlations can now be made and prognostic implications are already found. For instance, patients with AML carrying t(8;21), t(15;17) or inv(16) have a better prognosis for long-term survival. This is also illustrated by Figure 1, which shows data of the Dutch HOVON AML study. The definition of patients with a bad or good prognosis has already resulted in the adjustment of treatment protocols. In the near future, with the use of more defined molecular techniques, we might be able to characterize the chromosomal abnormality of each patient, to individualize his treatment and to recognize very early relapses.

Fluorescence in situ hybridization (FISH) and/or RNA-based polymerase chain reaction (RT-PCR) were used to analyze the breakpoints within the AML1 gene and the AML1 fusion transcripts in t(8;21) acute myeloid leukemia (AML). Twenty-two patients presented with the simple t(8;21)(q22;q22) and one with a complex variant t(8;2;16;21). In eight cases we used FISH with AML1 cosmid probes on metaphase chromosomes as well as RT-PCR to detect the junctions of MAL1/CDR (ETO,MTG8). Five cases were analyzed by FISH alone and ten cases by RT-PCR alone. By FISH we could identify three groups according to the distribution of the fluorescent signal. Signals were found in group 1 on chromosomes 21 and 21q+, in group 2 on chromosomes 21, 21q+ and 8q- and in group 3 on chromosomes 21 and 8q-. In all groups we could detect an identical AML1/CDR fusion transcript. This transcript showed splicing of AML1 exon 5 onto CDR. Thus regardless of the heterogeneity suggested by FISH, all the breakpoints in the AML1 gene were clustered in the same intro between exons 5 and 6. Our results bring to over one hundred the number of t(8;21) cases in which an identical translocation could be detected at molecular level by RT-PCR. The high sensitivity of the technique makes it suitable for the diagnosis of this translocation in different stages of the disease. The impact of the molecular detection of t(8;21) cells in clinical remission as far as the treatment and the management of the disease are concerned deserves further discussion.

Acute myeloid leukemic (AML) cells not only express receptors for various cytokines but also produce hemopoietic growth factors themselves. Thus, they are able to stimulate their own activation and proliferation, a phenomenon known as autocrine growth. The cell cycle kinetics of AML blast cells are susceptible to stimulation by a variety of cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-3, granulocyte colony stimulating factor and stem cell factor. GM-CSF and IL-3 can markedly enhance the cytotoxicity of chemotherapeutic agents by increasing the proportion of AML cells in S-phase at any given time and by altering the metabolic status of the AML cells. A number of clinical studies involving the use of GM-CSF in association with chemotherapy in patients with AML are currently ongoing. In the next few years the first clinical results will become available indicating whether the use of cytokines holds any benefit for patients with AML.