Randomized trial of daily versus weekly administration of 2-chlorodeoxyadenosine in patients with hairy cell leukemia: a multicenter phase III trial (SAKK 32/98).

Daily administration of 2-chlorodeoxyadenosine (Cladribine, CDA) is a standard treatment for hairy cell leukemia, but may cause severe neutropenia and neutropenic fever. This trial compared toxicity and efficacy of weekly versus daily CDA administration. One hundred patients were randomized to receive standard (CDA 0.14 mg/kg/day day 1-5 [Arm A]) or experimental treatment (CDA 0.14 mg/kg/day once weekly for 5 weeks [Arm B]). The primary endpoint was average leukocyte count within 6 weeks from randomization. Secondary endpoints included response rates, other acute hematotoxicity, acute infection rate, hospital admission, remission duration, event-free, and overall survival. There was no significant difference in average leukocyte count. Response rate (complete + partial remission) at week 10 was 78% (95% confidence interval (CI) 64-88%) in Arm A and 68% (95% CI 54-80%) in Arm B (p = 0.13). Best response rates during follow-up were identical (86%) in both arms. No significant difference was found in the rate of grade 3+4 leukocytopenia (94%vs. 84%), grade 3+4 neutropenia (90%vs. 80%), acute infection (44%vs. 40%), hospitalization (38%vs. 34%), and erythrocyte support (22%vs. 30%) within 10 weeks. Overall, these findings indicate that there are no apparent advantages in toxicity and efficacy by giving CDA weekly rather than daily.

DAPK2 is a novel E2F1/KLF6 target gene involved in their proapoptotic function.

Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca(2+)/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5'-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5' of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.

Cytogenetic response to prior treatment with interferon-alpha is predictive for survival after allogeneic hematopoietic stem cell transplantation in chronic myeloid leukemia.

We investigated the impact of a cytogenetic response (CyR) to IFN prior to and at the time of allogeneic hematopoietic stem cell transplantation (HSCT) on transplant-related mortality (TRM), relapse rate and survival probability after HSCT in 162 transplanted patients with chronic myeloid leukemia. One-hundred-one patients (62.3%) achieved a CyR prior to HSCT. Survival probabilities were higher in patients, who achieved any CyR prior to HSCT than in patients without CyR (63.6 vs 49.2%: P = 0.019). Survival probabilities in patients, who achieved a major CyR were better than in patients with minimal and minor CyR or in patients with no CyR (69.4 vs 58.8% vs 49.2%: P = 0.040). TRM and survival of chronic phase patients without CyR at the time of HSCT were similar to that of patients transplanted in advanced phase. Both groups combined had an outcome inferior to patients with at least minimal CyR (TRM, Gray test: P = 0.016, survival, log-rank test: P = 0.002). Univariate and multivariate analyses identified CyR prior to or at HSCT as a strong and independently favorable prognostic factor. We therefore conclude that allogeneic HSCT in CyR should be investigated prospectively as an alternative treatment option in defined patient groups.

Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1.

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), located on human chromosome 17p13.3, is frequently silenced in cancer by epigenetic mechanisms. Hypermethylated in cancer 1 belongs to the bric à brac/poxviruses and zinc-finger family of transcription factors and acts by repressing target gene expression. It has been shown that enforced p53 expression leads to increased HIC1 mRNA, and recent data suggest that p53 and Hic1 cooperate in tumorigenesis. In order to elucidate the regulation of HIC1 expression, we have analysed the HIC1 promoter region for p53-dependent induction of gene expression. Using progressively truncated luciferase reporter gene constructs, we have identified a p53-responsive element (PRE) 500 bp upstream of the TATA-box containing promoter P0 of HIC1, which is sequence specifically bound by p53 in vitro as assessed by electrophoretic mobility shift assays. We demonstrate that this HIC1 p53-responsive element (HIC1.PRE) is necessary and sufficient to mediate induction of transcription by p53. This result is supported by the observation that abolishing endogenous wild-type p53 function prevents HIC1 mRNA induction in response to UV-induced DNA damage. Other members of the p53 family, notably TAp73beta and DeltaNp63alpha, can also act through this HIC1.PRE to induce transcription of HIC1, and finally, hypermethylation of the HIC1 promoter attenuates inducibility by p53.

Implementation of the JACIE standards for a haematopoietic progenitor cell transplantation programme: a cost analysis.

The purpose of the present project was to analyse the costs incurred by the implementation of JACIE standards at a University Hospital with 1000 beds, performing some 40 autologous transplants per year. The cost analysis was performed on the basis of a prospective assessment of the time spent by all staff members involved with the implementation over a 14-month period of the quality management system (QMS) required by the JACIE standards. Two physicians worked on JACIE Section A (management=82 h), one physician and two nurses for section Ba (clinical unit adults=125.75 h), two physicians and three nurses for section Bp (clinical unit paediatrics=206 h), one physician, two nurses and one technician for section C (progenitor cell collection facility=105.75 h), and one physician and two technicians for section D (progenitor cell processing facility=426 h). The total time spent on the project amounted to 945.5 h with a total salary cost of \[euro]150 000. We concluded that implementation of the JACIE standards was accomplished within a 14-month period with a financial impact of approximately \[euro]150 000. The impact on quality parameters (eg clinical and laboratory end points, side effects) on HPC transplantation will be assessed in a second report after the first year of practical implementation.

Real-time process/quality control for HPC processing.

BACKGROUND: JACIE Standards (FACT Standards in the USA) have been implemented in Europe since 1999. An on-site accreditation inspection took place at our center in January 2004. The purpose of this work was to develop a real-time process/quality control system meeting the JACIE Standards for HPC release. METHODS: Data from 194 HPC processing procedures for autologous transplantation performed over a 5-year period were analyzed. The results of different processing methods applied at our facility were compared: (1) cryopreservation without washing cells (n=50), (2) washing cells (n=87), (3) cell-density separation (n=12) and (4) positive CD34 selection (n=45). RESULTS: Four critical control points were set for the validation of HPC processing: (a) number of lost CD34(+) cells during processing, (b) contamination, (c) viability of the cells after thawing and (d) ability to reconstitute hematopoiesis after transplantation. On the basis of statistical analysis, ranges of acceptable values were defined for each critical control point and for each processing method. Those acceptable values were used for cell release and real-time quality control. DISCUSSION: This study describes a model for the validation of HPC processing and for a real-time process/quality control system for HPC release. Optimization of processing techniques, standardization of methods and comparison between facilities will open the way towards external quality controls and quality improvement.

Treatment intensity significantly influencing fibrosis in bone marrow independently of the cytogenetic response: meta-analysis of the long-term results from two prospective controlled trials on chronic myeloid leukemia.

Bone marrow fibrosis (MF) has been shown to indicate therapy failure in Ph(+) chronic myeloid leukemia (CML). However, the results on the development of MF during interferon-alpha therapy of CML are controversial. The significance of the interferon dose has not been considered as yet. In total, 627 bone marrow biopsies taken prospectively from 200 patients with CML recruited in two studies using different doses of interferon-alpha +/- low-dose cytosine arabinoside were examined for MF before and during therapy. The results showed that the risk of MF depended significantly on the interferon-alpha dose applied (P<0.000005). MF progressed during low-dose therapy (3 x 5 x 10(6) IU/week), but was prevented from progression when applying high dose (5 x 10(6) IU/m(2)/per day). MF disappeared when high-dose interferon-alpha was combined with low-dose cytosine arabinoside (P<0.000005). The risk of death markedly increased when MF occurred or progressed (P<0.0009), independent of all other prognostic factors evaluated including the cytogenetic response. In conclusion, the effectiveness of interferon-alpha on MF depends on the treatment intensity. MF reverses when combining high-dose interferon-alpha with low-dose cytosine arabinoside, but progresses when applying low-dose interferon-alpha. MF appears to be a significant early indicator of ineffective therapy in CML.

[Myelodysplastic syndromes--an overview for the practitioner]. / Myelodysplastische Syndrome--gar nicht so selten.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematological diseases deriving from a clonally expanded precursor bone marrow cell. MDS are characterized by ineffective hematopoiesis, peripheral cytopenias of various degrees and increased risk for transformation into acute myeloid leukemia. The incidence is highest in elderly people. Diagnosis is based on morphology, exclusion of reactive causes of dyshematopoiesis and cytogenetics/FISH. Allogeneic stem cell transplantation, the only curative therapy so far, is not feasible for most of the patients. Other therapeutic options include supportive therapy such as blood transfusions and antibiotics, hematopoietic growth factors (rHuEPO, G-CSF), immunomodulating agents and chemotherapy. It is important to install iron chelation therapy in cases with long term transfusion dependency.

[Chronic myeloid leukemia--update]. / Chronische myeloische Leukämie--Update.

Chronic myeloid leukemia (CML) is a clonal stem cells disorder and belongs to the myeloproliferative diseases. Over the past decades seminal discoveries in the field of CML research have greatly contributed to our knowledge of leukemogenesis. The hallmark of the disease is the presence of the Philadelphia chromosome, the first described acquired non-random cytogenetic abnormality in human malignancies. This chromosomal abnormality is the result of a reciprocal translocation between chromosomes 9 and 22, t(9;22). At the molecular level this involves the fusion of the ABL protooncogene on chromosome 9 with the BCR (breakpoint cluster region) gene on chromosome 22. The fusion protein has increased tyrosine kinase activity and is a key event in the malignant transformation of a given progenitor cell in the bone marrow. Diagnosis of CML is based on the peripheral blood smear, bone marrow examination, the presence of the Philadelphia chromosome and its molecular correlate, the BCR-ABL transcript. Remarkable progress has been made in the treatment options over the last years which as a result rendered the therapeutic choices more complex and challenging. The current knowledge of treatment options is reviewed with particular emphasis on the newly introduced tyrosine kinase inhibitor Imatinib which opened an as yet unexpected promising avenue in the treatment of CML.

Marrow fibrosis, indicator of therapy failure in chronic myeloid leukemia - prospective long-term results from a randomized-controlled trial.

Marrow fibrosis (MF) has rarely been considered in therapy studies on chronic myeloid leukemia (CML), and there is a lack of long-term observations on the basis of sequential bone marrow biopsies (BMBs) taken prospectively during the course of disease. A total of 848 BMBs from 400 patients with Ph(+) CML recruited in the German randomized CML study I were examined for MF before and during therapy. In total, 110 patients had been randomized to receive interferon (IFN)-alpha, and 290 to receive chemotherapy (hydroxyurea (HU): 154, busulfan: 136). During IFN-alpha and HU medication, MF was reduced or did not increase for about 2 years. Evolving or progressive MF was an independent and early predictor of therapy failure about 2 years earlier than indicated by changes in the peripheral blood, spleen size, marrow blast count and cytogenetics (P<0.00005), resulting in a significant shortening of the survival times of patients independent of the type of therapy applied including allografting (multivariate analyses; P<0.00005). The analyzed long-term observations strongly indicate that MF is an independent poor prognostic complication of CML, allowing an early prediction of therapy failure. Consideration of the fiber content in marrow may therefore significantly improve the prediction of therapy efficacy and outcome of disease.

Acquired von Willebrand syndrome in a myeloproliferative disorder. Case 6.

We present a woman (age: 57 years) with an excessive bleeding episode under acetylsalicylic acid after bone marrow puncture due to an acquired von Willebrand syndrome (avWS) in the context of a myeloproliferative disorder. The laboratory features showed a high platelet concentration and a qualitative defect of von Willebrand factor (vWF) with a low normal vWF ristocetin cofactor activity, a normal vWF antigen and a decrease of the larger vWF multimers in plasma. The exact mechanism of avWS is still incompletely resolved. Myeloproliferative diseases are one of several underlying disorders that may cause avWS. The diagnosis of the underlying disease is important because its treatment may lead to an improvement of the vWF abnormality. For symptomatic treatment of bleeding, desmopressin, vWF concentrate infusion, intravenous immunoglobulin and/or fibrinolysis inhibitors can be tried.

Randomized comparison of interferon alpha and hydroxyurea with hydroxyurea monotherapy in chronic myeloid leukemia (CML-study II): prolongation of survival by the combination of interferon alpha and hydroxyurea.

The optimum treatment conditions of interferon (IFN) alpha therapy in chronic myeloid leukemia (CML) are still controversial. To evaluate the role of hydroxyurea (HU) for the outcome of IFN therapy, we conducted a randomized trial to compare the combination of IFN and HU vs HU monotherapy (CML-study II). From February 1991 to December 1994, 376 patients with newly diagnosed CML in chronic phase were randomized. In all, 340 patients were Ph/BCR-ABL positive and evaluable. Randomization was unbalanced 1:2 in favor of the combination therapy, since study conditions were identical to the previous CML-study I and it had been planned in advance to add the HU patients of study I (n=194) to the HU control group. Therefore, a total of 534 patients were evaluable (226 patients with IFN/HU and 308 patients with HU). Analyses were according to intention-to-treat. Median observation time of nontransplanted living patients was 7.6 years (7.9 years for IFN/HU and 7.3 years for HU). The risk profile (new CML score) was available for 532 patients: 200 patients (38%) were low, 239 patients (45%) intermediate, and 93 patients (17%) high risk. Complete hematologic response rates were higher in IFN/HU-treated patients (59 vs 32%). Of 169 evaluable IFN/HU-treated patients (75%), 104 patients (62%) achieved a cytogenetic response that was complete in 12% (n=21), major in 14% (n=24), and at least minimal in 35% (n=59). Of the 534 patients, 105 (20%) underwent allogeneic stem cell transplantation in first chronic phase. In the low-risk group, 65 of 200 patients were transplanted (33%), 30 (13%) in the intermediate-risk group, and nine (10%) in the high-risk group. Duration of chronic phase was 55 months for IFN/HU and 41 months for HU (P<0.0001). Median survival was 64 months for IFN/HU and 53 months for HU-treated patients (P=0.0063). We conclude that IFN in combination with HU achieves a significant long-term survival advantage over HU monotherapy. In view of the data of CML-study I, these results suggest that IFN/HU is also superior to IFN alone. HU should be combined with IFN in IFN-based therapies and for comparisons with new therapies.

Treatment of hairy cell leukemia with cladribine (2-chlorodeoxyadenosine) by subcutaneous bolus injection: a phase II study.

BACKGROUND: To assess the activity and toxicity of 2-chlorodeoxyadenosine (cladribine, CDA) given by subcutaneous bolus injections to patients with hairy cell leukemia (HCL). PATIENTS AND METHODS: Sixty-two eligible patients with classic or prolymphocytic HCL (33 non-pretreated patients, 15 patients with relapse after previous treatment, and 14 patients with progressive disease during a treatment other than CDA) were treated with CDA 0.14 mg/kg/day by subcutaneous bolus injections for five consecutive days. Response status was repeatedly assessed according to the Consensus Resolution criteria. RESULTS: Complete and partial remissions were seen in 47 (76%) and 13 (21%) patients, respectively, for a response rate of 97%. All responses were achieved with a single treatment course. Most responses occurred early (i.e. within 10 weeks) after start of CDA therapy, but response quality improved during weeks and even months after treatment completion. The median time to treatment failure for all patients was 38 months. Leukopenia was the main toxicity. Granulocyte nadir (median 0.2 x 10(9)/l) was strongly associated with the incidence of infections (P = 0.0013). Non-specific lymphopenia occurred early after CDA treatment, and normal lymphocytes recovered slowly over several months. No significant associations were found between infections and nadir count of lymphocytes or any lymphocyte subpopulation. No opportunistic infections were observed. CONCLUSIONS: One course of CDA given by subcutaneous bolus injections is very effective in HCL. The subcutaneous administration is more convenient for patients and care providers, and has a similar toxicity profile to continuous intravenous infusion. The subcutaneous administration of CDA is a substantial improvement and should be offered to every patient with HCL requiring treatment with CDA.

Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53.

Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the N-terminal transactivation domain (Delta Np73) was described in mice. In this study, we report the cloning and characterisation of the human Delta Np73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human Delta Np73 lacks the transactivation domain and starts with an alternative exon (exon 3'). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, Delta Np73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the Delta Np73 is strongly up-regulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of Delta Np73 is exerted through a p53 responsive element located on the Delta N promoter. Expression of Delta Np73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of Delta Np73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of Delta Np73 in primary tumours would clarify this issue.

Expression of TCL1 in hematologic disorders.

OBJECTIVE: TCL1, MTCP1 and TCL1b are three members of a new family of oncogenes that are expressed in T cell leukemias of ataxia telangiectasia patients (T-PLL, T-CLL). TCL1 is located at 14q32.1 and activated by juxtaposition to the alpha/delta-locus at 14q11 or beta-locus at 7q35 of the T cell receptor during the reciprocal translocations t(14;14)(q11;q32), t(7;14)(q35;q32), or inversion inv(14)(q11;q32). TCL1 encodes a predominantly cytoplasmic protein of 114 aa (14 kD) of unknown function. Recent studies suggest that TCL1 promotes cell survival rather than stimulating cell proliferation, as previously proposed. METHODS: In an attempt to clarify the contexts in which TCL1 is expressed, we investigated TCL1 expression in 114 lymphoma and leukemia patients by Northern blot, RT-PCR and immunohistochemistry. RESULTS: TCL1 expression is restricted to lymphoid cells, and is found in neoplastic (T and B cell neoplasms, and Hodgkin's disease) and nonneoplastic proliferations (reactive lesions). Out of 114 cases, 18 neoplasms of myeloid and 4 cases of epithelial origin were TCL1-negative. In lesions of the lymphoid system, both low- and high-grade lymphomas were found to express TCL1. CONCLUSIONS: We propose that TCL1 expression especially in high-grade B cell non-Hodgkin's lymphomas might interfere with B cell differentiation and promote the transition from low- to high-grade lymphoma.

Different p16INK4a and p14ARF expression patterns in acute myeloid leukaemia and normal blood leukocytes.

The p16INK4a gene is often disrupted or transcriptionally silenced by CpG island methylation in human cancers. However, in acute myeloid leukaemia (AML) alterations of the INK4a-ARF tumour suppressor locus are rarely found despite the noted variable p16INK4a mRNA and protein levels. The p14ARF, an alternative reading frame protein encoded from the same INK4a-ARF locus, is a potent tumour suppressor functionally linked to p53. There is little known regarding the role of p14ARF in primary human tumours. Therefore, we analysed the expression patterns of these two tumour suppressors in 37 cases of AML. The relative expression of p16INK4a and p14ARF mRNA in AML blasts, measured by a specific p16INK4a/p14ARF multiplex RT-PCR, was significantly shifted towards p14ARF whereas relatively lower levels of p16INK4a were detected. Quantitative RT-PCR revealed significantly higher expression of both transcripts in AML blasts when compared to normal differentiated myeloid cells or CD34+ progenitor cells. Furthermore, a good correlation between p16INK4a protein and mRNA was observed, whereas no correlation was found with p14ARF. Our results suggest: a) increased levels of both p16INK4a and p14ARF may participate in the pathogenesis of AML, b) that high p14ARF mRNA expression might influence p16INK4a transcription and c) that post-transcriptional regulatory mechanisms are important for p14ARF expression.

Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia.

The p73 protein shares structural and functional similarities with the tumour-suppressor p53, but its role in neoplastic transformation is unknown. Alternative splicing leads to the expression of at least nine p73 C-terminal mRNA splice variants (alpha, beta, gamma, delta, epsilon, zeta, eta, eta1, theta). In this survey, we analyse the expression of p73 by real-time quantitative RT-PCR, its known C-terminal variants with an RT-PCR-Southern technique and by Western blot in samples of 51 patients with B-CLL, normal B lymphocytes from eight individuals, and five haematopoetic cell lines. p73alpha protein expression positively correlated with higher risk B-CLL stages (P = 0.046). Total p73 mRNA expression was higher (P = 0.01) and p73alpha protein more frequently detected (P = 0.008) in B-CLL compared with normal CD19+-B-lymphocytes. p73 C-terminal mRNA variants were expressed both in B-CLL and in normal B-lymphocytes, but their expression was biased since the gamma (P = 0.041), the theta (P < 0.001), and the eta variant (P = 0.033) prevailed in normal B-lymphocytes. In summary, we conclude that the accumulation of p73, the expression pattern of particular p73 variants and its link to progression may play a distinct role in the molecular pathology B-CLL.