Diagnosed Childhood Acute Lymphoblastic Leukemia Research Articles

Analysis of multicenter efficacy of acute lymphoblastic leukemia in older children

To retrospectively analyze the clinical characteristics and the treatment outcomes of older children with acute lymphoblastic leukemia (ALL), and to evaluate the multicenter cooperation regimen (ALL-2005). The clinical data of 103 newly diagnosed ALL children aged 10 to 18 years old from five hospitals were enrolled in this study. They were all received ALL-2005 protocol. The clinical characteristics, the event-free survival (EFS), the overall survival (OS) and the prognostic analysis were evaluated. (1) Of the 103 patients, 62 were boys and 41 girls, with a median age of 12.3 years old. According to immunophenotyping, 90 (87.4% ) of 103 patients were diagnosed as B-ALL and 13 (12.6%) as T-ALL. According to risk factor, 65 (63.1%) were in intermediate risk group (MR-ALL) and 38 (36.9%) in high risk group (HR-ALL). Central nervous system leukemia (CNSL) happened in 4 (3.9%) patients at diagnosis. Of the 89 patients received chromosome test, 58 (65.2%) obtained the test results, including 21(36.2%) with aberrational chromosomes and 37 (63.8%) with normal karyotype. Of 81 patients received molecular biological test, 16 (19.8%) were positive for fusion gene. (2) After induction therapy, 97 (94.2%) obtained complete remission (CR). Twenty-eight patients relapsed with a median time of 11.9 months (ranged 2.9-57.8 months), and 38 (36.9%) patients died during the treatment. As of September 30, 2012, the median follow-up was 47 months (ranged 0.4-92.6 months). The 5-year EFS and 5-year OS of ALL patients were (60.2 ± 4.8)% and(64.1 ± 4.7)%. The 5-year EFS of MR-ALL and HR-ALL were (73.8 ± 5.5)% and (31.6 ± 8.3)% (P<0.01), the 5-year OS of MR- ALL and HR-ALL were (78.5 ± 5.1)% and (35.9 ± 8.0)% (P<0.01), respectively. (3) Cox proportion hazard regression model analysis indicated that age of 14-18 years old and BCR- ABL translocation or t(9;22) were independent risk prognostic factor for 5-year EFS. The incidence and prognosis in older childhood ALL were related with age, risk and biological characteristics. BCR-ABL translocation or t(9;22) was the risk factor of prognosis. ALL- 2005 protocol was recommended as the regimen for older childhood ALL.

Outcome of Tumor Lysis Syndrome with Hydration and Alkalinization in Children with Acute Lymphoblastic Leukemia

Objective: To observe the out come of tumor lysis syndrome (TLS) following treatment with hydration and alkalinization in children with acute lymphoblastic leukemia.Methodology: This is an observational study which included 30 diagnosed ALL children who were at high risk of developing TLS in the Department of Pediatric Hematology &amp; Oncology, BSMMU, from January 2010 to July 2010. Result: The mean (±SD) age was found 10.2±2.9 years and maximum (46.7%) numbers were found between 11 to 15 years. Male female ratio was 2.3:1. High risk patients for developing TLS presented with huge organomegaly such as hepatomegaly, splenomegaly, and lymphadenopathy associated with fever, anemia, and bony tenderness. For diagnosis of Acute Lymphoblastic Leukemia (ALL) bone marrow study was done in all patients and immunophenotyping in 40.0% patients. Hydration, Alkalinization, Tab Allopurinol and Oral Aluminum hydroxide were used in all study patients while treatment. WBC and serum uric acid were found higher in all patients during baseline investigation. After hydration and Alkalinization serum potassium, serum phosphate, serum calcium and serum creatinine level were normal within 72 hours and serum uric acid within day five. Mortality was found 6.7% and the cause of mortality was septicemia. DOI: http://dx.doi.org/10.3329/bjms.v11i4.12598 Bangladesh Journal of Medical Science Vol. 11 No. 04 Oct’12

Overweight as a Prognostic Factor in Children With Acute Lymphoblastic Leukemia

Our purpose was to investigate the prognostic impact of overweight/obesity in 5-year event-free survival (EFS) in a cohort of children with acute lymphoblastic leukemia (ALL). We retrospectively analyzed 181 newly diagnosed ALL children enrolled between 1990 and 2009 and treated with Berlin-Frankfurt-Munich (BFM) protocols. The majority of children in our cohort were <10 years-old. Our data clearly indicated that overweight/obesity is an independent predictor of relapse risk, mainly in the intermediate- and high-risk groups (HR) of children. These results could be explained by changes in the chemotherapy pharmacokinetics in overweight/obese patients and by the antiapoptotic effects in leukemic cells caused by adipocytes.

New markers for minimal residual disease detection in acute lymphoblastic leukemia

AbstractTo identify new markers for minimal residual disease (MRD) detection in acute lymphoblastic leukemia (ALL), we compared genome-wide gene expression of lymphoblasts from 270 patients with newly diagnosed childhood ALL to that of normal CD19+CD10+ B-cell progenitors (n = 4). Expression of 30 genes differentially expressed by ≥ 3-fold in at least 25% of cases of ALL (or 40% of ALL subtypes) was tested by flow cytometry in 200 B-lineage ALL and 61 nonleukemic BM samples, including samples containing hematogones. Of the 30 markers, 22 (CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, CD49f) were differentially expressed in up to 81.4% of ALL cases; expression of some markers was associated with the presence of genetic abnormalities. Results of MRD detection by flow cytometry with these markers correlated well with those of molecular testing (52 follow-up samples from 18 patients); sequential studies during treatment and diagnosis-relapse comparisons documented their stability. When incorporated in 6-marker combinations, the new markers afforded the detection of 1 leukemic cell among 105 BM cells. These new markers should allow MRD studies in all B-lineage ALL patients, and substantially improve their sensitivity.

Soluble Fas and Fas ligand and prognosis in children with acute lymphoblastic leukemia

The soluble forms of Fas and its ligand (sFas and sFasL) correlate with disease progression in various malignancies. We compared serum levels of sFas and sFasL in children with acute lymphoblastic leukemia and healthy children to determine the prognostic significance of these molecules. Serum levels of sFas and sFasL were measured with an enzyme-linked immunosorbent assay in 48 patients with newly diagnosed childhood acute lymphoblastic leukemia and 38 healthy children. Cut-off values of sFas and sFasL levels were based on their levels in controls. Clinical and laboratory characteristics were recorded on admission. The mean serum concentration of sFas was 243 ± 40 pg/mL in patients and 238 ± 29 pg/mL in controls. Serum levels of sFasL were 4.33 ± 0.25 ng/mL in patients and 4.27 ± 0.11 ng/mL in controls. Neither difference was significant. Based on the cut-off value, 12.5% of the patients were positive for sFas, and 16.6% were positive for sFasL. Survival was significantly longer in sFasL-positive patients (394 ± 69.6 vs. 254 ± 24.3 days) and the duration of complete remission was also longer (380 ± 65.0 vs. 246 ± 26.0 days) than in sFasL-negative patients (P < 0.02), indicating the important role of this molecule in the response to therapy. Higher sFas levels were associated with hepatosplenomegaly (P < 0.047). In conclusion, sFasL positivity was associated with a favorable outcome in ALL patients.

Relapse of Acute Lymphoblastic Leukemia in Children in the Context of Microarray Analyses

Over the last four decades the treatment of patients with newly diagnosed childhood acute lymphoblastic leukemia (ALL) has improved remarkably. However, still about 20% of children with ALL relapse despite risk-adapted polychemotherapy. The prognosis of relapsed ALL is relatively poor, even with modern aggressive chemotherapy. Identification of the biological and genetic mechanisms contributing to recurrence in patients with ALL is critical for the development of effective therapeutic strategies to treat refractory leukemic patients. Allogeneic hematopoietic stem-cell transplantation is the treatment of choice for many children with relapsed ALL. The gene expression profile obtained by microarray technology could provide important determinants of the drug response and clinical outcome in childhood ALL. Incorporation of the data on expression levels of newly identified genes into existing strategies of risk stratification might improve clinical management. Current microarray data show correlation of in vitro drug resistance with significant patterns of gene expression and explain clinical differences between early and late relapse. Genes involved in cell proliferation, self-renewal and differentiation, protein biosynthesis, carbohydrate metabolism, and DNA replication and repair are usually among those highly expressed in relapsed lymphoblasts. Current status and future perspectives of microarray data on gene expression and drug resistance profile in relapsed pediatric ALL are discussed in this review.

Glycogen synthase kinase-3β inhibition induces nuclear factor-κB-mediated apoptosis in pediatric acute lymphocyte leukemia cells

BackgroundMolecular therapies that target genetic abnormalities in leukemic cells and their affected signaling pathways have been emerging in pediatric acute lymphoblastic leukemia (ALL). Glycogen synthase kinase-3β (GSK-3β) has recently been found to positively regulate the activity of nuclear factor-κB (NF-κB). Here, we investigated the relationship between GSK-3β inhibition and NF-κB in apoptosis of pediatric primary leukemia cells obtained from 39 newly diagnosed ALL children in China.MethodsBone marrow mononuclear cells (BMMC) were isolated by density gradient centrifugation from the heparinized aspirates of children with ALL. We used immunofluorescence staining to detect nuclear GSK-3β in these cells. After treatment with chemically distinct GSK-3β inhibitors in vitro, NF-κB transcriptional activity was identified by means of western blotting and electrophoretic mobility shift assay (EMSA). NF-κB-mediated apoptosis was detected by Annexin V-PE/7-AAD double-staining flow cytometry. The expression level of the survivin gene was detected by reverse-transcriptase polymerase chain reaction (RT-PCR).ResultsGSK-3β significantly accumulates in the nuclei of ALL cells than in the nuclei of control cells. Cell death induced by GSK-3β inhibition in ALL cells was mediated by a downregulation of NF-κB p65 transcriptional activity. GSK-3β inhibition significantly decreased the expression of the NF-κB target gene survivin.ConclusionsThese results indicate that inhibition of GSK-3β downregulates the NF-κB activation pathway, leading to suppression of the expression of an NF-κB-regulated gene and promotion of apoptosis in ALL cells in vitro. Furthermore, our findings suggest that GSK-3β or NF-κB is a potential therapeutic target in the treatment of pediatric ALL.

Prospective bone ultrasound patterns during childhood acute lymphoblastic leukemia treatment

Objective Bone impairment is a well-known complication in childhood acute lymphoblastic leukemia (ALL) survivors but less is known about bone dynamics during ALL therapy. We longitudinally assessed by Quantitative Ultrasound (QUS) skeletal modifications during this treatment. Materials and methods Forty-four newly diagnosed ALL children underwent bone measurement by QUS parameters BTT (Bone Transmission Time) and AD-SoS (Amplitude-Dependent Speed of Sound), mainly reliant on bone density and cortical thickness, respectively. Measurements were performed at diagnosis, and 6, 12, and 24 months thereafter. The occurrence of skeletal complications such as fractures, vertebral collapse, osteonecrosis, and osteopenia was related to measurement outcome. Results A rapid deterioration of bone properties measured by BTT and AD-SoS was evident in the first semester of therapy ( p < 0.001). Subsequently, the next measurements were characterized by progressive uncoupling of the two QUS parameters ( p < 0.001). These were both significantly reduced at the end of therapy ( p < 0.001). Twelve subjects with in-treatment skeletal complications displayed an almost two-fold decrease of both parameters ( p < 0.001). BTT decreasing more than 1 Standard Deviation (SD) over 6 months of therapy was able to predict skeletal complication occurrence ( p < 0.001). Conclusion This report represents the largest longitudinal cohort systematically submitted to bone condition assessment from the beginning to the end of therapy for childhood ALL. Bone deterioration occurs early and persists throughout therapy, consistent with bone properties uncoupling. This pattern possibly reflects an initial impairment of both mineral density and cortical thickness with a subsequent recovery of this latter. QUS permits an early detection of bone deterioration and related skeletal complications in childhood ALL.

Detection of minimal residual disease in childhood acute lymphoblastic leukemia by multi-parameter flow cytometry

Objective To establish a flow cytometric measurement of detecting minimal residual disease(MRD) according to the leukemia-associated immunophenotypes in children with acute lymphoblastic leukemia(ALL) and to explore the significance of MRD detection in ALL children for a individualized treatment. Methods A variety of four-color fluorescent antibody combinations were used to investigate the children's normal bone marrow. The normal bone marrow pattern at two-parameter plots was established to identify the residual tumor cells, seventy-five bone marrow samples from newly diagnosed ALL children were analyzed with four-color cytometry to determined the optimal combinations which can clearly distinguish the tumor cells from normal cells. The bone marrow samples were monitored with the combination panel in 60 patients at the end of induction therapy and follow-up treatment. Cytomorphology test, PCR amplification of 29 fusion genes as well as IgG and TCR gene rearrangements were performed simultaneously. Results Sixty-nine cases (92.0%) could be identified for effective antibody combinations to monitor MRD by four-color cytometry. Fusion genes or IgG and T cell receptor (TCR) gene rearrangements can be detected in 21 cases (28.0%) to monitor MRD by PCR. No MRD can be detected in 25 bone marrow samples at the end of induction therapy and follow-up treatment. Four-color cytometry could detect as low as 0.021%-4.130% residual leukemia cells. Conclusion MRD can be monitored by flow cytometry which is faster than PCR, and the sensitivity is superior to morphology method. Key words: Precursor cell lymphoblastic leukemia-lymphoma; Neoplasm; residual; Flow cytometry

Pharmacokinetic, pharmacodynamic and intracellular effects of PEG-asparaginase in newly diagnosed childhood acute lymphoblastic leukemia: results from a single agent window study

L-asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is thought to result from depletion of asparagine in serum and cells. We investigated the clinical response in vivo of 1000 IU/m(2) pegylated (PEG)-asparaginase and its pharmacokinetic, pharmacodynamic and intracellular effects in children with newly diagnosed ALL before start of combination chemotherapy. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common/pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (P=0.01) and BCRABL-positive ALL (P=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-asparaginase (P=0.02) and both were prognostic factors for long-term event-free survival (hazard ratio 6.4, P=0.004; hazard ratio 3.7, P=0.01). After administration of one in vivo dose of PEG-asparaginase no changes in apoptotic parameters or in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. One additional dose of PEG-asparaginase upfront ALL treatment did not lead to other severe toxicities.

Garlic compounds selectively kill childhood pre-B acute lymphoblastic leukemia cells in vitro without reducing T-cell function: Potential therapeutic use in the treatment of ALL

Drugs used for remission induction therapy for childhood precursor-B acute lymphoblastic leukemia (ALL) are nonselective for malignant cells. Several garlic compounds have been shown to induce apoptosis of cancer cells and to alter lymphocyte function. To investigate the effect of garlic on the apoptosis of ALL cells and lymphocyte immune function, cells from newly diagnosed childhood ALL patients were cultured with several commonly used chemotherapeutic agents and several garlic compounds. Apoptosis, lymphocyte proliferation and T-cell cytokine production were determined using multiparameter flow cytometry. At concentrations of garlic compounds that did not result in significant increases in Annexin V and 7-AAD staining of normal lymphocytes, there was a significant increase in apoptosis of ALL cells with no alteration of T-cell proliferation as determined by CD25/CD69 upregulation or interferongamma, interleukin-2 or tumor necrosis factor-alpha intracellular cytokine production. In contrast, the presence of chemotherapeutic agents resulted in nonselective increases in both lymphocyte and ALL apoptosis and a decrease in T-cell proliferation and cytokine production. In conclusion, we show selective apoptosis of malignant cells by garlic compounds that do not alter T-cell immune function and indicate the potential therapeutic benefit of garlic compounds in the treatment of childhood ALL.

Pharmacokinetic and Pharmacodynamic Effects of PEG-Asparaginase in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: Results from a Single Agent Window Study.

L-Asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia. The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Several studies have shown that in vitro resistance to this drug is an independent prognostic factor in ALL. We investigated the clinical response of one in vivo dose of 1000 IU/m2 PEG-Asparaginase and its pharmacokinetic and pharmacodynamic effects in children with newly diagnosed ALL before the start of combination chemotherapy. 57 children (36M / 21F) were enrolled in the study: 2 pro B-ALL, 38 common/ pre B-ALL and 17 T-ALL. Genotyping of precursor B-ALL revealed 11 hyperdiploid, 8 TELAML1 positive, 2 BCRABL positive, no MLL rearrangement, 8 normal, 11 others. The clinical response to PEG-Asparaginase on day 0 (5 days after the PEG-Asparaginase infusion) was defined as good when the number of leukemic cells of peripheral blood was < 1 × 109/L, as intermediate when leukemic cells were 1-10 × 109/L, and as poor when leukemic cells were > 10 × 109/L. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common / pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (p=0.01). Both BCRABL positive ALL cases showed a poor response (p=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-Asparaginase (p=0.02) and both in vitro as well as in vivo response were prognostic factors for long-term event-free survival (Hazard ratio 6.4; p=0.004, and Hazard ratio 3.7; p=0.01, respectively). The L-Asparaginase activity in the serum was >100 IU/L for at least 15 days. The asparagine levels remained below the detection limit of 0.2 mM for at least 26 days with a concomitant rise in serum aspartate and glutamate. These findings confirm that PEG-Asparaginase will yield its pharmacodynamic effects for 2-4 weeks. After administration of one in vivo dose of 1000 IU/m2 PEG-Asparaginase no changes in apoptotic parameters or changes in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. Otherwise it is possible that in vivo mesenchymal cells from the bone marrow supply leukemic blasts with asparagine in response to treatment with L-Asparaginase.Conclusion: The clinical response to one dose of 1000 IU/m2 PEG-Asparaginase intravenously is related to phenotype and genotype and predicts outcome. These results suggest that children with ALL with a poor clinical response to PEG-Asparaginase might benefit from a more intensive antileukemic therapy.

In vivo control of acute lymphoblastic leukemia by immunostimulatory CpG oligonucleotides

AbstractDespite considerable success in treating newly diagnosed childhood acute lymphoblastic leukemia (ALL), relapsed disease remains a significant clinical challenge. Using a NOD/SCID mouse xenograft model, we report that immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODNs) stimulate significant immune activity against primary human ALL cells in vivo. The administration of CpG ODNs induced a significant reduction in systemic leukemia burden, mediated continued disease control, and significantly improved survival of mice with established human ALL. The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODNs and correlated with the production of IL-12p70, IFN-α, and IFN-γ by the host. In addition, depletion of natural killer cells by anti–asialo-GM1 treatment significantly reduced the in vivo antileukemic activity of CpG ODN. This antileukemia effect was not limited to the xenograft model because natural killer cell–dependent killing of ALL by human peripheral blood mononuclear cells (PBMCs) was also increased by CpG ODN stimulation. These results suggest that CpG ODNs have potential as therapeutic agents for the treatment of ALL.

Oxidative Status and Its Contribution to Extracellular Cytokine Secretion in Children with Acute Lymphoblastic Leukemia.

Recent data suggest that oxidative-antioxidative imbalance may influence cytokine secretion in children with acute lymphoblastic leukemia (ALL). Plasma cytokines concentrations are reported to have an effect on clinical course of the patients with lymphoid malignancies. The aim of the study was analysis of oxidative status (Malonylodialdehyde, MDA) and antioxidant defense (Superoxide dismutase, SOD; glutathione peroxidase, GPX; total antioxidant status, TAS and vitamin E) in peripheral blood and evaluation of plasma concentration of IL-2, IL-4, IL-10 and TNF-alpha in children with acute lymphoblastic leukemia.Material and methods: Study group consisted of 23 newly diagnosed ALL children, including 13 boys and 10 girls, with median age 5 years (range, 0.5–16). Control group consisted of 21 healthy children (11 boys and 7 girls) with median age 7 years (range, 2–17). TAS, SOD, GPX status were estimated using commercially available Randox Laboratories Ltd kits. Vitamin E and MDA plasma concentrations were measured spectrofluorimetrically. Cytokine concentrations were measured by quantitative immunoassay tests: IL-2 and IL-4 plasma levels were assessed using Bender MedSystems test (Vienna, Austria), IL-10 and TNF-alpha by R&D system tests. All assays were performed twice: on diagnosis and 6 weeks thereafter.Results: GPX activity in study group was significantly higher before and during treatment than in control subjects (1.5-fold, p=0.02 and 2-fold, p=0.0007, respectively). MDA concentrations were higher in ALL group before treatment (1.4-fold, p=0.0001) and 6 weeks thereafter (1.2 fold, p=0.01) than in control group. SOD activity, TAS and vitamin E concentrations did not differ between the groups. IL-10 level was found to be significantly increased on ALL diagnosis, but not during therapy, when compared to control group (2-fold, p=0.02). TNF-alpha level was higher in patients before treatment then during treatment (3-fold, p=0.02) and as compare to the controls (2,5-fold, ns). IL-2 and IL-4 plasma concentrations were comparable in all groups in both time points. There was a correlation in children with ALL after 6 weeks therapy between MDA and IL-10 concentrations (r=0.59, p<0,05) as well as MDA and TNF-alpha concentrations (r=0,58, p<0,05). In control group MDA was correlated with IL-10(r=0.59, p<0.05). Median follow-up of the study group is 23 months, and at that time relapses occurred in 3 out of 23 patients. 2-fold decrease in median GPX were observed in patients, who relapsed afterward,. Median IL-10 plasma concentrations in children, who subsequently relapsed were 4-fold higher at diagnosis and 8-fold higher after 6 weeks of therapy then in patients who are still in remission (despite of achieving complete remission by all study group at 6th week of treatment).Conclusions: Oxidation process in plasma of children with ALL is significantly increased on diagnosis. Pro-oxidative status may contribute to IL-10 and TNF secretion and affect efficacy of treatment.

P-glycoprotein 170 expression and function as an adverse independent prognostic factor in childhood acute lymphoblastic leukemia

Little is known about the prognostic role of multidrug resistance (MDR) in newly diagnosed childhood acute lymphoblastic leukemia (ALL). P-glycoprotein 170 (MDR1), a cellular drug efflux pump, is thought to be one of the major causes of MDR. The aim of this retrospective study was to evaluate in 85 children with ALL the impact of the MDR1 product of the mdr-1 gene on the achievement of complete remission (CR) and outcome. MDR1 protein expression was performed by immunocytochemistry (ICC), and flow cytometry (FC). MDR1 functional activity was performed by a rhodamine (Rhd)-123 efflux test with or without verapamil. All patients enrolled in our study were treated with AIEOP ALL 91-95 protocols. At diagnosis, 40 patients (47%) expressed MDR1 protein at significant levels, and 45 (53%) were MDR1 negative. Forty-three of the latter patients were also negative for MDR1 function, while 34/40 (85%) patients MDR1 positive preserved the function. Rhd-123 efflux was inhibited by the MDR modulator verapamil in 12/40 (30%) patients. After induction treatment, CR was achieved in 77/85 children (90.6%). All patients who did not achieve CR were MDR1 positive. Twenty-nine patients relapsed, 17 (58.6%) of whom were MDR1 positive. The 10-year overall survival (OS) rate, and disease-free survival (DFS) for MDR1 negative patients compared to MDR1 positive patients were 75.7% versus 54.8%, and 67.5% versus 46%, respectively. The 10-year event-free survival (EFS) rate was significantly higher (67.5% versus 36.8%) in the MDR1 negative group compared with the MDR1 positive population (p=0.001). Multivariate analysis showed that only EFS was independent of age, WBC count, immunophenotype, FAB subtype and prednisone response (p=0.019). Our results, derived from a monocentric study, demonstrate that MDR1 expression in childhood ALL is an independent adverse prognostic factor on outcome, and could be a useful biological marker of response in these patients. Moreover, MDR1 function was also a predictor of response, but only in univariate analysis.

Sensitization of Leukemic Cells for Glucocorticoids by Inhibition of NFκB Activity.

Based on their immunosuppressive and cytotoxic effects, glucocorticoids (GCs) find widespread use as immune-modulatory agents and in (childhood) leukemia, lymphomas and myelomas. Unfortunately, resistance to GCs is a major adverse prognostic factor and occurs in about 20% in newly diagnosed childhood acute lymphoblastic leukemia (ALL) and in more than 50% in relapsed ALL, while acute myeloid leukemia (AML) is largely unresponsive to GCs. In addition, adult AML is more often GC resistant compared to childhood AML. Recently, we observed (van der Heijden et al, Ann Rheum Dis 63: 131-137,2004) that prolonged exposure of human CEM-C7 T cell leukemia cells to the anti-rheumatic drug sulfasalazine (SSZ) sensitized these primary sensitive cells even further for GCs. SSZ is an inhibitor of activation of NFκB that is often therapeutically used in combination with methotrexate and prednisone. Following these observations, two human myeloid leukemia cell lines (THP-1 and U937) with inherent resistance to GCs (IC50 for prednisolone > 250 μM, IC50 dexamethasone > 25 μM) were exposed to SSZ to establish whether this would provoke GC sensitization. Indeed, prolonged SSZ exposure sensitized both myeloid leukemia cells for prednisolone (IC50 < 1 μM) and dexamethasone (IC50 < 0.05 μM). In order to elicit the mechanistic basis for the GC sensitizing effect of SSZ expression levels of Glucocorticoid Receptor α (GRα), NFκB p65 and Inhibitor of NFκB (IκBα) were analyzed. In contrast to GC-sensitive CEM-C7 cells, parental THP-1 and U937 cells had no detectable and constitutive expression of GRα and NFκB p65 protein, even though mRNA levels for both parameters were readily detectable. In SSZ-exposed THP-1 and U937 cells protein expression of NFκB p65 was markedly increased although it does not represent the active form of NFκB since protein expression of IκB increased concomitantly. Consistent with the sensitization for GCs in SSZ-exposed THP-1 and U937 cells was a marked increase in GRα protein expression, suggesting that GRα is post-transcriptionally prevented from degradation. In line with this concept is that co-incubation with a 26S proteasome inhibitor Bortezomib further enhanced (5-fold) GC sensitivity in SSZ-exposed THP-1 and U937 cells. Collectively, this study reveals that SSZ-exposure impairs activation of NFκB and facilitates stabilization of GRα protein, conditions which confer GC-sensitization in leukemic cells with primary resistance to GCs. In conclusion, modulation of GC resistance in acute leukemias by NFκB inhibitors is a novel, clinically attractive approach that warrants further preclinical and subsequent clinical investigations.

Newly diagnosed childhood acute lymphoblastic leukemia: update on prognostic factors and treatment.

The development of effective therapy for children with acute lymphoblastic leukemia is one of the great successes of clinical hematology andoncology. Fifty years ago, childhood acute lymphoblastic leukemia was universally fatal, but current long-term event-free survival rates are nearly 80%. Despite this improved outcome, there are still many challenges facing investigators today. In some recent clinical trials, the outcome of "high-risk" patients has approached that of "lower risk" patients, suggesting that currently applied clinical factors, such as age and presenting leukocyte count, no longer identify the 20% of newly diagnosed patients who ultimately will relapse. Additionally, therapy remains nonspecific, toxic, and sometimes lethal. As more children with acute lymphoblastic leukemia survive into adolescence and adulthood, there is a need to address the late sequelae of current therapy and to develop more leukemia-specific treatments. Promising avenues of research, which may identify biologically distinctive subsets of acute lymphoblastic leukemia and potential targets for novel therapies, include studies of minimal residual disease, lymphoblast genetics (including genetic profiling studies), and host-related pharmacogenomics.

Modification of topoisomerase genes copy number in newly diagnosed childhood acute lymphoblastic leukemia.

Topoisomerase genes were analyzed at both DNA and RNA levels in 25 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL). The results of molecular analysis were compared to risk group classification of children in order to identify molecular characteristics associated with response to therapy. At diagnosis, allelic imbalance at topo-isomerase IIalpha (TOP2A) gene locus was found in 75% of informative cases whereas topoisomerase I and IIbeta gene loci are altered in none or only one case, respectively. By semi-quantitative Polymerase chain reaction, we found a 2.5 to 8-fold TOP2A gene amplification in 72% of the children, which was correlated to gene overexpression in every case. These results show that TOP2A gene amplification is a frequent event in ALL at diagnosis. Interestingly, we also identified a small population of children that do not present TOP2A gene amplification or gene overexpression and who are significantly associated with very high risk classified patients showing glucocorticoid resistance. In conclusion, characterization of TOP2A gene status in childhood ALL at diagnosis provides useful complementary information for risk assessment.

Risk factors for traumatic and bloody lumbar puncture in children with acute lymphoblastic leukemia.

Traumatic or bloody lumbar puncture (LP) reduces the diagnostic value of the procedure and may worsen the outcome of patients with acute lymphoblastic leukemia (ALL). Little is known about the risk factors for traumatic and bloody LP. To determine the risk factors for traumatic and bloody LP. Retrospective cohort study of 956 consecutive patients with newly diagnosed childhood ALL who were treated at a pediatric cancer center between February 1984 and July 1998. All patients underwent a diagnostic LP followed by a median of 4 LPs to instill intrathecal chemotherapy. Traumatic LP was defined as an LP in which cerebrospinal fluid contained at least 10 red blood cells (RBCs) per microliter and bloody LP as one in which the cerebrospinal fluid contained at least 500 RBCs per microliter. Of the 5609 LPs evaluated, 1643 (29%) were traumatic and 581 (10%) were bloody. The estimated odds ratios (ORs) and 95% confidence intervals (CIs) for traumatic LP were 1.5 (95% CI, 1.2-1.8) for black vs white race, 2.3 (95% CI, 1.7-3.0) for age younger than 1 year vs 1 year or older, 1.4 (95% CI, 1.2-1.7) for early vs recent (dedicated procedure area and general anesthesia) treatment era, 1.5 (95% CI, 1.2-1.8) for platelet count of 100 x 10(3)/ microL or more vs less than 100 x 10(3 )/ microL, 10.8 (95% CI, 7.7-15.2) for short (1 day) vs longer (>15 days) interval since the previous LP, and 1.4 (95% CI, 1.1-1.8) for the least vs the most experienced practitioners. Analyses for bloody LP yielded similar results. The unmodifiable risk factors for traumatic and bloody LP include black race, age younger than 1 year, a traumatic or bloody previous LP performed within the past 2 weeks, and a previous LP performed when the platelet count was 50 x 10(3)/ microL or less. Modifiable risk factors include procedural factors reflected in treatment era, platelet count of 100 x 10(3)/ microL or less, an interval of 15 days or less between LPs, and a less experienced practitioner.

Expression of multidrug resistance-related genes does not contribute to risk factors in newly diagnosed childhood acute lymphoblastic leukemia

The present study was designed to evaluate the association of multidrug resistance gene (MDR1), multidrug resistance-associated protein (MRP) gene and lung resistance protein (LRP) gene expression (mRNA levels) with risk factors such as phenotype, age and leukocyte count in newly diagnosed childhood acute lymphoblastic leukemia, because biological mechanisms contributing to risk factors were not known. Expression of the MDR1, MRP and LRP genes in leukemic cells from 40 pediatric patients was quantitated using the reverse transcriptase polymerase chain reaction method. The associations of expression of these genes with age and leukocyte count, which are known as risk factors for acute lymphoblastic leukemia (ALL), were analyzed. Moreover, the associations with clinically classified risk-related groups (high-risk ALL and low-risk ALL) were also investigated. No positive correlations between expression of these genes and age or leukocyte count were found. The expression of these genes was not different among clinically classified risk-related groups. Expression (mRNA levels) of the MDR1, MRP and LRP genes did not contribute to risk factors in newly diagnosed childhood acute lymphoblastic leukemia.