Sabine Leiblein

High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML)

The search for molecular markers in AML that allow prediction of outcome has recently focused on genes involved in the regulation of programmed cell death (PCD). The aim of our study was to determine whether mRNA levels of Mdm-2, Bcl-2, Bcl-xL, Bad, and Bax are independent prognostic parameters for outcome. Transcript levels were analyzed by real-time quantitative RT-PCR in 232 samples collected either at diagnosis or following induction chemotherapy (ICT). Multivariate COX regression analysis adjusted for chemotherapy protocol, de novo vs secondary AML, and de novo vsrelapsed AML indicated: (1) At diagnosis, high expression of Bad (P = 0.015) and even more so high Bax and Bad levels (P = 0.018) predicted adverse outcome, regardless of the response to ICT. In patients who subsequently failed to enter complete remission (CR), high levels of Bad, Bax and Bax high/Bad high were associated with an increased relative risk (RR) to die from tumor (RR = 5.0 for Bad, 3.4 for Bax and 6.14 for Bax high/Bad high). (2) Following ICT, high expression of Bax (P = 0.005) and high Bcl-2/Bax ratios (P = 0.004) were independent predictors of unfavorable outcome, regardless of response to ICT. We conclude that high levels of Bax and Bad correlate with poor outcome, particularly in patients who do not enter CR and may serve as prognostic markers in AML.

FISH for BCR-ABL on interphases of peripheral blood neutrophils but not of unselected white cells correlates with bone marrow cytogenetics in CML patients treated with imatinib

Interphase fluorescence in situ hybridization (I-FISH) for the BCR-ABL translocation performed on peripheral blood (PB) white cells has been suggested as a surrogate for conventional bone marrow (BM) cytogenetics for monitoring patients with chronic myeloid leukemia (CML). I-FISH is faster, less costly, and does not require BM aspiration. For patients treated with interferon-alpha (IFN), a good correlation between the two methods has been demonstrated in several though not all studies. However, imatinib mesylate (STI571) has largely replaced IFN as the standard drug treatment for CML, raising the question if the results obtained in IFN-treated patients are applicable to patients on imatinib. We therefore compared the two methods in patients on imatinib and patients on other therapies, mainly IFN (collectively referred to as nonimatinib therapies). Our results demonstrate that the correlation between I-FISH and cytogenetics is much weaker in patients on imatinib than in patients on nonimatinib therapies. Correction of the I-FISH values for the proportion of lymphocytes barely improved the correlation, probably as a result of unpredictable proportions of Philadelphia-positive B cells. By contrast, I-FISH of PB neutrophils was much better correlated with BM cytogenetics. We conclude that I-FISH on unselected PB white cells is not suitable for monitoring patients on imatinib.

Monitoring of WT1 expression in PB and CD34 + donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning

Relapse of malignant disease remains the major complication in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) with reduced-intensity conditioning (RIC). In this study, we investigated the predictive value of disease-specific markers (DSMs), donor chimerism (DC) analysis of unsorted (UDC) or CD34+ sorted cells and Wilms’ tumor gene 1 (WT1) expression. Eighty-eight patients with AML or MDS were monitored after allogenic HCT following 2 Gy total-body irradiation with (n=84) or without (n=4) fludarabine 3 × 30 mg/m2, followed by cyclosporin A and mycophenolate mofetil. DSMs were determined by fluorescence in situ hybridization (FISH) and WT1 expression by real-time polymerase chain reaction. Chimerism analysis was performed on unsorted or CD34+ sorted cells, by FISH or short tandem repeat polymerase chain reaction. Twenty-one (24%) patients relapsed within 4 months after HCT. UDC, CD34+ DC and WT1 expression were each significant predictors of relapse with sensitivities ranging from 53 to 79% and specificities of 82–91%. Relapse within 28 days was excluded almost entirely on the basis of WT1 expression combined with CD34+ DC kinetics. Monitoring of WT1 expression and CD34+ DC predict relapse of AML and MDS after RIC-HCT.

Norepinephrine-Induced Changes in Cardiac Transforming Growth Factor-β Isoform Expression Pattern of Female and Male Rats

Transforming growth factor-&bgr; (TGF-&bgr;) is a ubiquitous growth-regulating protein with an essential role in tissue repair and formation of extracellular matrix (ECM). To better understand the role of different isoforms of TGF-&bgr; in the cardiac remodeling process induced by norepinephrine (NE), the expression of TGF-&bgr;1, TGF-&bgr;2, and TGF-&bgr;3 was studied and compared with the expression of collagen. NE (0.1 mg/kg · h) was intravenously infused in female and male Sprague-Dawley rats for several time periods, and freshly obtained ventricular myocardium after 1 day was dissociated into myocyte and nonmyocyte fractions. Prazosin (0.1 mg/kg · h) and metoprolol (1 mg/kg · h) were used to block &agr;- and &bgr;-adrenoceptors, respectively. After NE infusion, the three isoforms of TGF-&bgr; were differentially induced as far as the magnitude and the time course is concerned. The increased expression of TGF-&bgr;2 started earlier with a maximum after 12 hours and was more pronounced (10-fold elevation) than that of the other two isoforms, with a clear specificity for the left ventricle in female hearts. This specificity was also seen in male rats with 16-fold elevation of TGF-&bgr;2 after 1 day of NE-stimulation. The increase of TGF-&bgr;2 was significant only in the myocyte fraction obtained from female as well as from male hearts. The expression of the mRNA of all TGF-&bgr; isoforms of collagen type I and type III, and of the matrix metalloproteinase (MMP)-2 and its inhibitor TIMP-2 was reduced predominantly by &agr;-adrenoceptor blockade with prazosin. The increase in TGF-&bgr; isoforms correlated with that of the mRNA expression of collagens, MMP-2 and TIMP-2.

BCR-ABL transcripts are early predictors for hematological relapse in chronic myeloid leukemia after hematopoietic cell transplantation with reduced intensity conditioning

Kinetics of BCR-ABL transcript elimination and its prognostic implications on relapse were analyzed in patients with chronic myeloid leukemia (CML) after reduced intensity hematopoietic cell transplantation (HCT). In all, 19 CML patients were conditioned with 2 Gy total-body irradiation in combination with (n=14) or without (n=3) fludarabine 3 × 30 mg/m2 (Flu) or 4.5 Gy total lymphoid irradiation (TLI) with Flu and OKT3 3 × 5 mg (n=2) and were treated with cyclosporine (CSP) and mycophenolate mofetil after allogeneic HCT. BCR-ABL transcripts were analyzed by nested RT-PCR and Taqman® RT-PCR on days +28, +56 and +84 after HCT and were evaluated for their association with relapse. Of the 19 patients, 14 achieved sustained remissions of which six had a negative RT-PCR 28 days after HCT. Five patients relapsed +41, +54, +57, +136 and +234 days after HCT. Predictors for relapse were advanced disease stage (P=0.02) and slow reduction of BCR-ABL transcripts at day 28 (P=0.006) and day 56 (P=0.047) post-transplant. We conclude that a complete clearance of BCR-ABL transcripts is achievable within 4 weeks from HCT even after minimal conditioning and that early kinetics of BCR-ABL transcripts significantly correlate with the probability of hematological relapse.

The impact of the age of HLA-identical siblings on mobilization and collection of PBSCs for allogeneic hematopoietic cell transplantation

With the increasing age of patients undergoing allogeneic hematopoietic cell transplantation (HCT), the age of matched related sibling donors (MRDs) is expected to increase. Donor safety and the impact of donors’ age on mobilization, collection of peripheral hematopoietic progenitor cells (HPCs), subsequent engraftment and the incidence of GVHD were retrospectively analyzed. A total of 167 patients received HCT from an MRD. Median donors’ age was 48 years (67 (40%) donors were ⩾50 years including 34 donors ⩾60 years). Side effects under mobilization and apheresis were age independent. Grafts from donors <50 years contained more CD34+ cells (median 9 × 106/kg recipients body weight (RBW)) compared with older donors (median 5.9 × 106/kg RBW) (P<0.0005), whereas harvests from donors ⩾60 years contained more natural killer (NK) cells (P=0.003). Engraftment occurred at a median of 12 days after HCT irrespective of donors’ age. Increasing age of MRD did not preclude successful mobilization, collection of HPC and engraftment. In the context of more NK cells in grafts from elderly donors, the impact of donors’ age on outcome after HCT warrants further studies. Although short-term toxicities of apheresis were not increased with increasing age, long-term donor safety remains an important issue.

Absolute Levels of MDR-1, MRP, and BCL-2 MRNA and Tumor Remission in Acute Leukemia

Mononuclear cells prepared from peripheral blood or bone marrow of 119 AML and 28 ALL patients prior and following therapy were analyzed for absolute transcript levels of the chemoresistance genes mdr-1 and MRP, and the proto-oncogene bcl-2, by validated contamination-protected quantitative RT-PCR. In newly diagnosed AML mainly tumors of the granulocytic lineage (FAB M1-M2) expressed increased mdr-1 mRNA amounts. The MRP gene was expressed in all investigated samples without relation to a particular FAB class. High initial expression of both genes did not confer a poor prognosis even at high number of CD34+ cells. Data compared prior to and after therapy start (paired samples) revealed that AML patients who did not respond to therapy (NR) expressed increased levels of mdr-1 mRNA, as well as MRP and bcl-2 cDNA normalized to GAPDH reference transcripts, when compared to patients achieving complete remission (CR; p = 0.003, 0.008 and 0.0005, respectively). In ALL-NR the mdr-1 and bcl-2 genes were entirely more active after induction chemotherapy. Arbitrary cut-off values were established in order to delimit pathological from non-pathological gene expression. 59% of studied AML and 33% of ALL-NR exceeded the arbitrary values (mdr-1: > 2 amol/microgram RNA, MRP: > 10 zmol/amol GAPDH, bcl-2: > 5 zmol/amol GAPDH) for one and 11% of AML-NR for two parameters. Only 17% of the AML-CR and none of the ALL-CR group were above these limits. The results indicate that high individual activity of usually one, rarely two of the investigated genes might be associated with poor clinical outcome in treated acute leukemia.

Highly Elevated Serum Hepcidin in Patients with Acute Myeloid Leukemia prior to and after Allogeneic Hematopoietic Cell Transplantation: Does This Protect from Excessive Parenchymal Iron Loading?

Hepcidin is upregulated by inflammation and iron. Inherited (HFE genotype) and treatment-related factors (blood units (BU), Iron overload) affecting hepcidin (measured by C-ELISA) were studied in 42 consecutive patients with AML prior to and after allogeneic hematopoietic cell transplantation (HCT). Results. Elevated serum ferritin pre- and post-HCT was present in all patients. Median hepcidin pre- and post-HCT of 358 and 398 ng/mL, respectively, were elevated compared to controls (median 52 ng/mL) (P < .0001). Liver and renal function, prior chemotherapies, and conditioning had no impact on hepcidin. Despite higher total BU after HCT compared to pretransplantation (P < .0005), pre- and posttransplant ferritin and hepcidin were similar. BU influenced ferritin (P = .001) and hepcidin (P = .001). No correlation of pre- or posttransplant hepcidin with pretransplant ferritin was found. HFE genotype did not influence hepcidin. Conclusions. Hepcidin is elevated in AML patients pre- and post-HCT due to transfusional iron-loading suggesting that hepcidin synthesis remains intact despite chemotherapy and HCT.

Low tumor burden is associated with early B-cell reconstitution and is a predictor of favorable outcome after non-myeloablative stem cell transplant for chronic lymphocytic leukemia

Abstract Reconstitution, engraftment kinetics and tumor cell clearance were analyzed after reduced intensity conditioning hematopoietic cell transplant (RIC-HCT) in patients with chronic lymphocytic leukemia (CLL). Patients were transplanted from unrelated (n = 40) or related (n = 10) donors after fludarabine and 2 Gy total body irradiation followed by cyclosporine and mycophenolate mofetil. The vast majority of patients (96%) engrafted with absolute neutrophil count (ANC) > 0.5 × 109/L at day + 22. CLL cells decreased (median 2%, range 0–69%) within 28 days, but disappeared by day + 180 after HCT. Donor T-cell chimerism increased to > 95% at day 56 and donor B-cell chimerism to 94% at day + 360. Overall survival was 51 ± 8%, incidence of progression 37 ± 7% and non-relapse related mortality (NRM) 30 ± 7% at 4 years. The most common causes of NRM were graft-versus-host disease (GvHD) (14%) and sepsis (6%). Disease status at HCT was significantly associated with early B-cell reconstitution (p = 0.04) and with increased risk of relapse/progression in univariate and multivariate analysis (p = 0.022). Tumor cells were undetectable by day + 180, although B-cell reconstitution did not occur until 1.5 years after RIC-HCT. The best predictors for progression-free survival (PFS) and overall survival (OS) were complete response (CR) or first partial response (PR1) and the absence of bulky disease at transplant, respectively.

Chemotherapy for mobilisation of Ph-negative progenitor cells from patients with CML: impact of different mobilisation regimens

Mobilised peripheral blood stem cells are widely used for autografting in patients with chronic myeloid leukaemia (CML) and it is generally thought that a high proportion of Ph-negative progenitor cells in the graft is desirable. We report here the results of 91 stem cell mobilisations performed with various chemotherapy regimens followed by G-CSF. We show that mobilisation of Ph-negative cells is possible after diagnosis as well as in advanced stages of the disease. The yield of Ph-negative cells is highly dependent on the chemotherapy regimen: while the combination of idarubicin and cytarabin for 3–5 days (IC3–5) mobilised Ph-negative cells in most patients, high-dose cyclophosphamide was ineffective. Mobilisation of Ph-negative progenitor cells after IC3 was at least as effective as after IC5; however, less apheresis sessions were required, and toxicity was much reduced after IC3. Compared to historical controls, IC was equally effective as the widely used ICE/minilCE (idarubicin, cytarabin, etoposide) protocol. No correlation was found between graft quality and the cytogenetic response to subsequent treatment with interferon-α. We conclude that IC3 is an effective and well-tolerated regimen for mobilising Ph-negative cells that compares well with more aggressive approaches such as IC5 and ICE/minilCE. Bone Marrow Transplantation (2001) 27, 1125–1132.