ALBERT

Beschreibung: Congenital dyserythropoietic anemia type II (CDA II) is the most frequent type of congenital dyserythropoietic anemia. More than 200 cases have been described, but with the exception of a report by the International CDA II Registry, these reports include only small numbers of cases and no data on the lifetime evolution of the disease. Since 1967, we were able to follow 48 cases of CDA II from 43 families for up to 35 years. All patients exhibit chronic anemia of variable severity requiring regular red cell transfusions only in a minority of children; 60% developed gallstones before the age of 30 years, and 16 patients had cholecystectomy between 8 and 34 years of age. Iron overload was a frequent complication. In 16 cases, iron depletion started between 7 and 36 years. Three patients died from secondary hemochromatosis. Splenectomy, performed in 22 cases, led to moderate increases in hemoglobin values and eliminated the need for transfusions but did not prevent further iron loading. The current recommendation is to consider splenectomy if the anemia compromises patients' performance, and to manage iron overload according to the guidelines derived from patients with thalassemia. (Blood. 2003;102:4576-4581)

Beschreibung: In 65 patients with hemophagocytic lymphohistiocytosis (HLH), we found an as yet undescribed heterogeneity of defects in cellular cytotoxicity when assay conditions were modified by the incubation time, the presence of mitogen, or interleukin-2 (IL-2). The standard 4-hour natural killer (NK) test against K562 targets was negative in all patients. In patients deficient in type 1 (n = 21), type 2 (n = 5), and type 4 (n = 8) HLH, negative NK function could be reconstituted by mitogen, by IL-2, or by prolongation of the incubation time (16 hours), respectively. Most patients (n = 31) displayed the type 3 defect, defined by a lack of any cellular cytotoxicity independent of assay variations. The characteristic hypercytokinemia also concerned counterregulatory cytokines, such as proinflammatory interferon-γ (IFN-γ), simultaneously elevated with suppressive IL-10 in 38% of types 1–, 2–, and 4–deficient patients and in 71% of type 3–deficient patients. Elevated IFN-γ alone correlated with high liver enzymes, but sCD95-ligand and sCD25 did not—though these markers were expected to indicate the extent of histiocytic organ infiltration. Outcome analysis revealed more deaths in patients with type 3 deficiency (P = .017). Molecular defects were associated with homozygously mutated perforin only in 4 patients, but other type 3 patients expressed normal transcripts of effector molecules for target-cell apoptosis, including perforin and granzyme family members, as demonstrated by RNase protection analysis. Thus, target-cell recognition or differentiation defects are likely to explain this severe phenotype in HLH. Hyperactive phagocytes combined with NK defects may imply defects on the level of the antigen-presenting cell.

Beschreibung: ICL670 (deferasirox) is an investigational once-daily oral iron chelator that has demonstrated the ability to induce sustained, clinically relevant reductions in liver iron content (LIC) in heavily transfused patients with β-thalassemia and iron overload. The efficacy and safety of ICL670 is being assessed in a multicenter, randomized, open-label Phase III study in comparison with deferoxamine (DFO) in patients aged ≥2 years with β-thalassemia and transfusional hemosiderosis. Between March and November 2003, 587 patients began treatment (296 on ICL670; 291 on DFO) in the following 12 countries : Italy (200), Turkey (87), Tunisia (68), US (48), Greece (46), Germany (27), Argentina (24), Belgium (24), Brazil (20), UK (18), Canada (13) and France (12). Based on LIC at baseline (2–3, 〉3–7, 〉7–14 and 〉14 mg Fe/g dw), patients were randomized in a 1:1 ratio to receive either oral ICL670 once daily at doses of 5, 10, 20 or 30 mg/kg, respectively, or subcutaneous DFO at doses of 20–60 mg/kg/day for 5 days/week. Treatment was for one year initially, to be followed by an extension phase during which patients randomized to DFO may switch to ICL670. LIC, the primary outcome variable, was assessed at baseline by liver biopsy or, in some children, non-invasively by magnetic susceptometry using a Superconducting QUantum Interference Device (SQUID). LIC will be reassessed after 12 months of therapy in each patient using the same methodology as at baseline. Liver biopsies are analyzed at a single center (Rennes, France) and SQUID assessments are performed in 3 centers (Turin, Italy; Hamburg, Germany; Oakland, US). At baseline, median (25–75th percentiles) LIC was 13.0 mg Fe/g dw (7.2–21.0) by biopsy and 5.6 (4.0–7.7) in those patients assessed by SQUID. Total body iron balance will be assessed to determine the relative chelation efficacies of ICL670 and DFO. A summary of patient demographics and baseline characteristics (median values or no. of pts) is given in the table. ICL670 has been well tolerated with mild, transient gastrointestinal complaints as the main AEs with a suspected relationship to study drug. As of May 2004, 8 patients on ICL670 and 2 on DFO had discontinued therapy due to AEs. The key efficacy and safety data from the initial 12 months of therapy for all randomized patients will be available late November 2004. Treatment group (by initial dose) ICL670 (n=296) Deferoxamine (n=291) 10 mg/kg n = ≤ 94 20 mg/kg n = 83 30 mg/kg n = 119

Beschreibung: Children with T-lineage Acute Lymphoblastic Leukemia (T-ALL) have a higher relapse-risk and are in-vitro more resistant to therapeutic drugs compared to ALL patients with a precursor-B phenotype. Cellular resistance to anti-cancer agents has previously shown to be associated with failure of P53 family member signaling by abrogation of P53 function due to loss-of-function mutations or dominant-negative inhibition by isoforms of P73 lacking (part of) the N-terminal transactivation domain (P73ΔEX2, P73ΔEX2/3, ΔN-P73 and ΔN’-P73). Since p53 mutations are not commonly found in T-ALL, we investigated the expression levels of p73 splice variants in relation to drug resistance in children with T-ALL. Splice variants were quantitatively measured at the mRNA level in leukemic cells of 55 T-ALL patients and mononuclear cells of 12 non-leukemic controls. TA-p73 (transactivation competent), p73Δex2, p73Δex2/3, ΔN-p73 and ΔN’-p73 were all found to be present at a relatively higher mRNA level in T-ALL patients than controls (P 〈 0.05 for all), suggesting that expression of the TP73 gene is deregulated in T-ALL. Resistance of T-ALL cells to the DNA damaging drug daunorubicin correlated with mRNA levels of the dominant-negative variants of p73, i.e. ΔN-p73 and ΔN’-p73 (Rs = 0.38, P = 0.03). In contrast, expression of none of the variants, including ΔN-p73 and ΔN’-p73, was related to resistance of T-ALL cells to non-DNA damaging drugs (prednisolone, vincristine and L-asparaginase). In conclusion, high expression of ΔN-p73 and ΔN’-p73 variants possibly contributes to resistance to DNA damaging drugs in childhood T-ALL.

Beschreibung: Previous studies on apoptosis defects in acute lymphoblastic leukemia (ALL) have focused on chemotherapy-induced, primarily mitochondrial death pathways. Yet, immunologic surveillance mechanisms including sensitization to apoptotic signals mediated via the death receptor CD95 might contribute to leukemic control. Here, we show that primary B-cell precursor ALL cells from children escape from receptor-dependent cell death in 2 ways: Resting ALL blasts are protected from receptor-mediated apoptosis due to the absence of CD95 surface expression. However, even though CD40 ligation results in up-regulation of CD95, ALL blasts, unlike normal B cells, remain resistant to apoptosis. We show that this apoptosis resistance involves the selective up-regulation of the short isoforms of the caspase-8 inhibitor c-FLIP acting directly at the CD95 receptor level. Treatment with cycloheximide during CD40 activation prevents up-regulation of those c-FLIP isoforms and sensitizes ALL cells toward CD95-mediated apoptosis. We therefore propose that induction of the short c-FLIP isoforms inhibits the onset of CD95-induced apoptosis in primary CD40-stimulated ALL cells despite high CD95 expression.

Beschreibung: The German cooperative study group for childhood acute lymphoblastic leukemia (COALL-92) was designed to examine the clinical effectiveness of thioguanine (TG) versus mercaptopurine (MP) in maintenance treatment of childhood acute lymphoblastic leukemia (ALL) in a randomized multicenter trial. TG and MP are prodrugs and have to be converted intracellularly to 6-thioguanine nucleotides (TGNs) for cytostatic activity. TG is converted into TGN in fewer steps and has been shown to be more cytotoxic in equimolar doses in vitro compared with 6-MP. Therefore, a higher effectiveness of TG in maintenance treatment was postulated. Of 521 patients enrolled into the protocol, 474 were randomized to receive either MP or TG during maintenance therapy in a daily oral dose. After a median observation time of 6.6 years, the probability of event-free survival was 79% ± 3% for the MP group (238 children) and 78% ± 3% in the TG group (236 patients). In spite of TGN levels, exceeding those of the MP group 7 times, treatment with TG did not improve the outcome but was more complicated to handle due to a specific toxicity profile of prolonged myelosuppression with marked thrombocytopenia. Therefore, MP should remain the preferred drug for maintenance treatment of ALL, unless other studies demonstrate superiority of TG in larger trials or selected patient groups.

Beschreibung: Resistance of leukemic cells to chemotherapeutic agents is associated with an unfavorable outcome in pediatric acute lymphoblastic leukemia (ALL). To investigate the underlying mechanisms of cellular drug resistance, the activation of various apoptotic parameters in leukemic cells from 50 children with ALL was studied after in vitro exposure with 4 important drugs in ALL therapy (prednisolone, vincristine, l-asparaginase, and daunorubicin). Exposure to each drug resulted in early induction of phosphatidylserine (PS) externalization and mitochondrial transmembrane (Δψm) depolarization followed by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) inactivation in the majority of patients. For all 4 drugs, a significant inverse correlation was found between cellular drug resistance and (1) the percentage of cells with PS externalization (〈 .001 〈 P 〈 .008) and (2) the percentage of cells with Δψm depolarization (.002 〈 P 〈 .02). However, the percentage of cells with caspase-3 activation and the percentage of cells with PARP inactivation showed a significant inverse correlation with cellular resistance for prednisolone (P = .001; P = .001) and l-asparaginase (P = .01; P = .001) only. This suggests that caspase-3 activation and PARP inactivation are not essential for vincristine- and daunorubicin-induced apoptosis. In conclusion, resistance to 4 unrelated drugs is associated with defect(s) upstream or at the level of PS externalization and Δψm depolarization. This leads to decreased activation of apoptotic parameters in resistant cases of pediatric ALL. (Blood. 2003;102:4541-4546)

Beschreibung: Drug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7, -8, -10, and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) in cells from children with acute lymphoblastic leukemia (ALL; n = 43) and acute myeloid leukemia (AML; n = 10). PARP expression was present in all B-lineage samples, but absent in 4 of 15 T-lineage ALL samples and 3 of 10 AML cases, which was not caused by genomic deletions. PARP expression was a median 7-fold lower in T-lineage ALL (P 〈 .001) and 10-fold lower in AML (P 〈 .001) compared with B-lineage ALL. PARP expression was 4-fold lower in prednisolone, vincristine and L-asparaginase (PVA)-resistant compared with PVA-sensitive ALL patients (P 〈 .001). Procaspase-2 expression was 3-fold lower in T-lineage ALL (P = .022) and AML (P = .014) compared with B-lineage ALL. In addition, procaspase-2 expression was 2-fold lower in PVA-resistant compared to PVA-sensitive ALL patients (P = .042). No relation between apoptotic protease-activating factor 1 (Apaf-1), procaspases-3, -6, -7, -8, -10, and drug resistance was found. In conclusion, low baseline expression of PARP and procaspase-2 is related to cellular drug resistance in childhood acute lymphoblastic leukemia. (Blood. 2005;106:1817-1823)

Beschreibung: The influence of the microenvironment on the activation status and behaviour of ALL blasts is critical for interactions with the immune system in vivo. The capacity of B cells to respond to CD40-ligand (CD40L) stimulation is critical for their sensitisation to immunological control mechanisms and susceptibility to apoptotic signals. Primary precursor B-ALL blasts (BCP-ALL; n=32) lack CD95-expression (mean±SE; 4.2±0.6% positive cells) and are resistant to apoptosis while significant up-regulation of CD95 is apparent upon CD40-stimulation in BCP-ALL blasts that reaches a plateau after 72 h. Yet, in spite of equivalent CD95-upregulation in ALL blasts (58.3±6.5%; n=17) and normal B cells (59.3±13.1%) specific apoptosis is markedly lower in ALL compared to mature B cells (19.1±3% vs 36.7±5.5%). Resistance to apoptosis in ALL blasts and its reversibility after cycloheximid treatment suggest that anti-apoptotic mechanisms prevent induction of cell death via CD95 ligation in CD40 activated blasts. In accordance, in CD40-activated ALL blasts caspase 8 and 3 activity is not enhanced upon CD95 ligation in contrast to an 1.8±0.3 and 1.7±0.3 fold increase in caspase activity in stimulated normal B cells (n=7), suggesting a block of the apoptotic cascade in BCP-ALLrelatively close to the receptor level. CD40L-activated ALL blasts and normal B cells were submitted to western blot analysis with respect to the molecules associated to the death-inducing signalling complex (DISC). FADD and the zymogen form of caspase-8 are constitutively expressed in both malignant and non malignant B cells with no modulation following CD40 ligation. In contrast, the anti-apoptotic short isoform of the c-FLICE inhibitory protein FLIPS is weakly expressed in naïve blasts and B cells, but strongly up-regulated upon 72h CD40-ligation in ALL with only barely detectable levels in CD40-activated normal B cells. We therefore propose, that prolonged induction of the FLIPS expression inhibits the onset of apoptosis despite high CD95 surface expression levels in BCP-ALL blasts. As an additional anti-apoptotic mechanism inhibiting the downstream effector caspases we demonstrated significant upregulation of the inhibitor of apoptosis protein (IAP) survivin in CD40-activated BCP-ALL (n=6) compared to the unstimulated control (632pg/ml±200pg/ml vs 180pg/ml±52pg/ml). Thus, we identified FLIPS as a CD40-regulated upstream anti-apoptotic element and concomitant downstream upregulation of survivin protein expression as critical mechanisms contributing to blast cell resistance to apoptosis.

Beschreibung: T-lineage ALL is an unfavorable subtype of childhood ALL and is associated with in-vitro resistance to drugs. Therefore the identification of novel genes that may serve as targets to modulate drug resistance is desirable. We compared the gene expression profile of 28 pediatric T-ALL patients being either sensitive (DNRS) or resistant (DNRR) to daunorubicin. The aryl hydrocarbon receptor (AHR) gene appeared to be highly discriminating between DNRS and DNRR T-ALL patients. AHR is known to mediate signal transduction in response to xenobiotics by activating the transcription of xenobiotic-responsive genes such as CYP1A1 and CYP1A2. Expression analysis by real-time quantitative PCR confirmed that basal AHR mRNA levels in ALL cells derived from patients is correlated with DNR resistance (Rs=0.41, P=0.02). Exposure to DNR of the REH cell line expressing a low AHR level led to a 40-fold induction of AHR mRNA. In two other cell lines (i.e. HL60 and SEMK-2) expressing high levels of AHR the upregulation was only 1.4-fold. In REH, the 40-fold induction of AHR after DNR exposure was inhibited by 40% after pre-exposure to the AHR inhibitors salicylamide (SAL) and geldanamycin (GA). Pre-incubation of leukemic cells of T-ALL patients with SAL or GA prior to DNR exposure had a synergistic effect on DNR sensitivity. In addition, transfection of SEMK-2 cells with AHR-specific siRNA resulted in the reduction of AHR expression by 80% after 24 h and had also a synergistic effect on DNR induced cell kill up to 96 hours after siRNA treatment. We conclude that a high expression of the AHR gene is involved in DNR resistance in childhood T-ALL and that AHR may serve as a very attractive new therapeutic target.