Martin Weisser

Reduced-intensity conditioning using TBI (8 Gy), fludarabine, cyclophosphamide and ATG in elderly CML patients provides excellent results especially when performed in the early course of the disease.

Summary:Allogeneic bone marrow or stem cell transplantation is a curative therapeutic option for chronic myelogenous leukemia. In order to decrease the toxicity of the procedure, the dosage of total body irradiation was reduced from 12 to 8 Gy and subsequently the dose of cyclophosphamide from 120 to 80 mg/kg. The purine analogue fludarabine, ATG, cyclosporine A and a short course of methotrexate were given for immune suppression. So far, 35 elderly CML patients with sibling and unrelated donors have been transplanted. Transplant-related mortality at day +100 was 11%. After engraftment, all patients achieved a complete cytogenetic remission. Relapse occurred in 14% of the patients. The risk of relapse was significantly higher in those patients transplanted in second chronic or accelerated phase (P=0.048). After a median follow-up of 30 months (range 12–62), 63% of the patients are alive. Those patients transplanted within the first year from diagnosis had an overall survival of 79% (P=0.049), emphasizing the benefit of early transplantation. Stepwise reduction of conditioning intensity resulted in stable engraftment, low relapse rates and encouraging overall survival in this high-risk patient group.

Allogeneic stem-cell transplantation provides excellent results in advanced stage chronic myeloid leukemia with major cytogenetic response to pre-transplant imatinib therapy.

To determine the impact of imatinib therapy prior to allogeneic stem-cell transplantation in advanced stage chronic myelogenous leukaemia (CML), 30 CML patients who had received imatinib as part of pre-transplant treatment were analysed, with special emphasis on the cytogenetic response to imatinib therapy shortly before transplantation. Median patient age was 51 years (range, 24 – 64). At the time of transplantation all patients were in second or higher chronic phase (CP). The median follow-up after allogeneic transplantation was 360 days (range, 24 – 1524). Cox regression analysis revealed that the quality of cytogenetic response was a prognostic factor for transplant-related mortality (p = 0.050), relapse incidence (p = 0.015), leukaemia-free survival (p = 0.002) and overall survival (p = 0.006). A cytogenetic response with <35%BCR-ABL-positive interphase nuclei in FISH analysis from bone marrow was associated with a probability of overall survival of 81% at 3 years. In conclusion, our data suggest that advanced stage CML has an excellent outcome after allogeneic haematopoietic stem-cell transplantation when transplanted in the phase of cytogenetic response to imatinib.

Advanced age and high initial WBC influence the outcome of inv(3) (q21q26)/t(3;3) (q21;q26) positive AML.

AML with inv(3)/t(3;3) are generally considered of having a poor prognosis. For further insight in this rare entity the outcome of 65 inv(3)/t(3;3) positive AML cases were examined with special emphasis on patient and disease related factors at diagnosis. Survival data were available from 35 patients. A hematological CR was achieved in 16/35 patients (46%). Eight patients (50%) relapsed. The median duration of remission was 177 days. Probability of OS was 23% at 2 years. Advanced age and high initial WBC count were associated with shorter OS (p = 0.021 and p = 0.005, respectively). Loss of chromosome 7 was the most frequent additional aberration (n = 34; 52%), followed complex aberrant aberrations (n = 5). Cases with monosomy 7 or the presence of FLT3-length mutations (FLT3-LM)—detected in 13% of cases—were not associated with an even more inferior outcome. Allogeneic stem cell transplantation, performed in 12 cases, resulted in a probability of OS of 62% at 2 years. Our data (1) confirm that inv(3)/t(3;3) AML has a poor prognosis (2) show that age and initial WBC are risk factors for prognosis; (3) suggest that this group may benefit from allogeneic stem cell transplantation.

Reverse transcriptase-polymerase chain reaction based quantification of the combined MDS-EVI1/EVI1 gene in acute myeloid leukemia.

Molecular markers are increasingly important for the assessment of prognosis and for follow-up controls to detect minimal residual disease (MRD) in acute myeloid leukemia (AML). The protooncogene EVI1 (located in the chromosomal region 3q26) is involved in the pathogenesis of AML with 3q26 aberrations [1 – 3]. The exact mechanisms of leukemogenesis remain unclear. AML with 3q26 aberrations have often demonstrated abnormal transcriptional activity of the EVI1 gene. In addition, there have been reports of AML without 3q26 aberrations expressing EVI1 [4 – 6]. The present study aimed to establish a further follow-up marker for minimal residual disease in AML. Because the sensitivity of a polymerase chain reaction (PCR) assay is dependent on the level of initial expression level, we chose a PCR product spanning exons 7 – 10 that did not discriminate between the EVI1 and MDS-EVI1 gene to increase the percentage of samples with high expression levels. In a first step, the expression level of this combined MDS-EVI1/EVI1 transcript was assessed at primary diagnosis by real-time quantitative reverse transcriptase (RT)-PCR in separate cytogenetic subgroups of AML to determine AML subsets for which follow-up using this molecular marker might be especially useful. From 1997 to 2004, bone marrow samples of 424 patients were analysed at primary diagnosis. All cases were diagnosed as AML according to the WHO classification [7]. Cytogenetic analysis was performed centrally according to standard protocols. Cytogenetic data were classified according to the ISCN nomenclature [8]. In addition, bone marrow samples from 33 patients were sent in for follow-up assessment with a median follow-up sample number of four per patient (range 2 – 22). Mononuclear cells were obtained by Ficoll density gradient centrifugation. From each sample, mRNA of 56 10 human cells were isolated according to the MagnaPure.LC mRNA protocol for human cells (Roche Diagnostics, Mannheim, Germany) and eluted in 30 ml. Random primed cDNA synthesis with the use of Superscript II (Invitrogene, San Diego, CA, USA) was performed from diagnostic samples with 5 ml of mRNA corresponding to approximately 0.86 10 cells in a 50-ml reaction. From follow-up samples, all 30 ml of mRNA were transcribed into cDNA to gain a higher sensitivity. Quantitative PCR was performed using the LightCyclerSystem (Roche Diagnostics) applying hybridization probes as the detection format. PCR was performed using 2 ml mastermix (LightCycler Fast Start DNA Master Hybridization Probes, Roche Diagnostics), 4 mM MgCl2, 0.25 mM of each 30 and 50 fluorescent hybridization probe, 0.5 mM of each 30 and 50 primer (TibMolBiol, Berlin, Germany), 2 ml of cDNA and water to a final volume of 20 ml. Amplification occurred after initial incubation at 958C for 10 min in a three-step cycle procedure (denaturation 958C, 1 s, ramp rate 208C/s, annealing 648C, 10 s, ramp rate 208C/s, and extension 728C, 26 s, ramp rate 28C/s) for 45 cycles. Primers and hybridization probes to

Resistance to pretransplant imatinib therapy may adversely affect the outcome of allogeneic stem cell transplantation in CML

Resistance to pretransplant imatinib therapy may adversely affect the outcome of allogeneic stem cell transplantation in CML

The Choice of House Keeping Genes in MRD-Quantification of AML1-ETO Positive Acute Myeloid Leukemia

In modern hematology the quantification of minimal residual disease (MRD) during and after anti-leukemic therapy is of major interest. Reverse transcriptase polymerase chain reaction (RT-PCR) is the most sensitive and widely used method for detection and quantification of MRD. Common targets for MRD detection in acute myeloid leukemia (AML) are fusion transcripts, e.g., AML1-ETO, PML-RARα and CBFβ-MYH11, are specifically expressed by the leukemic clone (1). Several groups have detected molecular relapse of myeloid leukemias before hematologic manifestation by following ratios of fusion transcripts (2,3,4). Quantitative studies of RNA expression are usually performed relative to the expression of a housekeeping gene as an endogenous control that compensates for uneven RNA quality and different numbers of cells between samples. Housekeeping genes comprise a wide variety of genes that mainly code for proteins that are essential for cell function (5) and are assumed to be constitutively expressed in all cell types at relative stable levels. Recently, some reports have indicated that the expression of housekeeping genes can vary considerably depending on the experimental conditions (6,7). Other studies have shown that neoplastic cells may exhibit abnormal levels of housekeeping gene expression (8,9). Therefore, the choice of an appropriate housekeeping gene as an endogenous control is essential for the accuracy and reliability of a quantitative PCR approach. We have established a real-time RT-PCR protocol in our lab to quantify AML1-ETO fusion transcripts in t(8;21) positive AML patients. For this quantitative PCR approach, we examined the expression of four commonly used housekeeping genes — β-2-microglobulin (β2M), porphobilinogen deaminase (PBGD), glucose-6-phosphate dehydrogenase (G6PDH) and cABL — by realtime RT-PCR. Samples included bone marrow at primary diagnosis, follow-up, during and after cytostatic therapy, and the peripheral blood of healthy individuals.