Childhood Acute Lymphoblastic Leukemia Samples Research Articles

KIF11 serves as a cell cycle mediator in childhood acute lymphoblastic leukemia.

To identify key gene in childhood acute lymphoblastic leukemia (ALL) through weighted gene co-expression network analysis (WGCNA), and their enriched biological functions and signaling pathways. Array data of the GSE73578 dataset, involving 46 childhood ALL samples, were acquired from the Gene Expression Omnibus (GEO) database. Hub modules associated with childhood ALL were screened out by WGCNA. Enriched biological functions and signaling pathways were then identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Hub genes were selected by overlapping those between down-regulated genes in GSE73578, GSE4698 and the hub module. Guilt by association (GBA) was adopted to verify the function of the identified KIF11 gene and to predict its target genes. Regulatory effects of KIF11 on the proliferation and cell cycle progression of ALL in vitro were determined by cytological experiments. WGCNA showed that the yellow module was the most relevant to childhood ALL treatment, containing 698 genes that were enriched in cell division, mitotic nuclear division, DNA replication and DNA repair, cell cycle, DNA replication and the P53 signaling pathway. The KIF11 gene was screened out and predicted as a cell cycle mediator in childhood ALL. Knockdown of KIF11 in ALL cells inhibited cell proliferation and arrested cell cycle progression in G2/M phase. The KIF11 gene is critical in the treatment process of childhood ALL, which is a promising therapeutic target for childhood ALL.

ABERRANT PROMOTER HYPERMETHYLATION OF SELECTED APOPTOTIC GENES IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA AMONG NORTH INDIAN POPULATION

In present study, we investigated the prevalence of hypermethylation of caspase-8 (CASP8), TMS1 and DAPK genes in correlation with clinicopathological factors in childhood acute lymphoblastic leukemia (ALL). A case-control study has been conducted based on bone marrow and peripheral blood samples from 125 ALL patients and 100 sex-age matched healthy controls. Methylation specific polymerase chain reaction (PCR) and bisulfite sequencing PCR was performed to analyze the methylation status of these genes. Reverse transcription PCR and real time PCR was carried out to determine changes in the mRNA expression level of the genes due to hypermethylation. Hypermethylation of the 5´CpG islands of the CASP8, TMS1 and DAPK gene promoters was found in 3.2, 6.4, and 13.6% of 125 childhood ALL samples from north Indian population, respectively. There were significant differences in pattern of hypermethylation of TMS1 (p=0.045) and DAPK (p < 0.001) between patients and healthy controls. Down-regulation of mRNA expression was found in cases in which CASP8, TMS1 and DAPK were hypermethylated. The present study indicated the impact of hypermethylation-mediated inactivation of CASP8, TMS1 and DAPK genes, which is associated with risk of childhood ALL. This abnormality occurs in leukemogenesis and it may be used as a biomarker and for predicting the prognosis of ALL.

Targeted Inhibition of the MLL Transcriptional Complex By Proteosome Inhibitors Elicits a High Response Rate in Relapsed/Refractory MLL Rearranged Leukemia

Infants with MLL rearranged (MLLr) acute lymphoblastic leukemia (ALL) have a poor prognosis, with an event free survival of only 23-44%. Whole genome sequencing (WGS) of this subtype has revealed a paucity of cooperating mutations, with an average of 2.2 somatic single nucleotide variations and/or insertions/deletions per case. Despite recent progress in defining the epigenetic alterations that result from the expression of the MLL fusion protein, these insights have only recently begun to be extrapolated into the development of new therapeutic approaches whose benefits have yet to be defined. Thus, there remains an urgent need for the development of alternative approaches to improve outcomes in these patients.To identify compounds that are active in MLLr disease, we established in vitro and in vivo assays to evaluate drug sensitivity of primary infant ALL patient samples. 15 infant MLLr leukemia samples that have previously undergone WGS were xenografted into NOD/SCID/IL2Rγnull (NSG) mice. All samples engrafted and expanded in NSG mice, leading to overt leukemia with a latency of 49 to 276 days. Purification of leukemic blasts from a single moribund mouse yielded on average 108 cells, providing sufficient material to screen large numbers of compounds. In vitro conditions were defined that support growth in 40% of the patient specimens, allowing for a more accurate determination of drug sensitivity. Growth in vitro was associated with early onset of disease in NSG xenografts and younger age at presentation, allowing us to evaluate patient samples that represent aggressive high risk disease. Using this system, we tested bortezomib in addition to 28 other drugs, including standard ALL therapeutic agents as well as targeted kinase inhibitors and inhibitors of epigenetic marks. Three classes of agents were active in this system: anthracyclines, histone deacetylase inhibitors (HDACi), and the proteasome inhibitor bortezomib. In contrast to anthracyclines and HDACi, where IC50 values were on par with those reported in the literature for primary childhood ALL samples, MLLr infant samples required 10-100 fold less bortezomib to induce toxicity.Bortezomib has been shown to mediate responses through several mechanisms, including NFKB inhibition, stabilization of cell cycle regulatory proteins, and induction of apoptosis. Recently, proteasome inhibition has been demonstrated to lead to accumulated MLL fusion protein levels, triggering apoptosis and cell cycle arrest in MLLr cell lines. To determine if NFKB inhibition also plays a role, we evaluated cellular concentrations of the activated NFKB transcription factor, but failed to see decreased levels when MLLr cells were treated with bortezomib. Bortezomib has also been shown to deregulate ubiquitin stores and deplete histone H2B ubiquitination (H2Bub), an epigenetic mark that is linked to histone methylation and expression. Recently, several groups have demonstrated that H2Bub is required for DOT1L activity and HOX gene expression. We therefore evaluated H2Bub levels in bortezomib-treated patient samples and confirmed depletion of this epigenetic mark. Furthermore, patient samples treated with bortezomib downregulated both the MLL gene expression signature and signatures of downstream targets, such as cMYC, demonstrating that the MLL transcriptional program is inhibited in the presence of bortezomib. ChIP-seq is underway to map H2Bub and H3K79 methylation changes genome wide in response to treatment with bortezomib.The HDACi vorinostat and bortezomib have both been evaluated in Phase I and II pediatric leukemia clinical trials. Based on the safety and efficacy from these earlier studies, we treated 6 relapsed/refractory MLLr leukemia patients with a chemotherapy regimen that included mitoxantrone, vorinostat, and bortezomib. 4 patients had a complete response (CR), 1 patient had a partial response (PR) and 1 patient had stable disease for an overall response rate of 5/6 (83%). Clinical trials are in development to assess this combination further for both relapsed MLLr disease as well as newly diagnosed infant ALL. Our data suggests that these three classes of drugs, identified in our laboratory assays, are clinically active thus validating our system. We are now using this platform to proceed with a high throughput drug screen to identify additional compounds for future clinical development. DisclosuresOff Label Use: Vorinostat and Bortezomib for the treatment of pediatric leukemia.

PLK1 expression and BI 2536 effects in childhood acute lymphoblastic leukemia

Polo-like kinase 1 (PLK1) is a conserved kinase that mediates various mitotic events. Compelling data have repeatedly demonstrated its upregulation in different neoplasia, being frequently associated with poor prognosis. However, in childhood acute lymphoblastic leukemia (ALL), no studies have yet been conducted. PLK1 expression and association with biological features were evaluated in 65 consecutively diagnosed childhood ALL samples by quantitative real-time PCR. Moreover, the effects of a specific PLK1 inhibitor, BI 2536, was tested against a panel of nine ALL cell lines at nanomolar concentrations (10, 50, 100 nM). The mRNA expression of PLK1 showed great variability in pediatric ALL, but no difference was evidenced compared to normal bone marrow. Additionally, no association was found between PLK1 mRNA expression with any clinical or biological features. Alternatively, high mRNA expression of PLK1 was present in ALL cell lines. In vitro treatment with BI 2536 strongly diminished growth, while presenting significant reduction in colony formation capacity and increased apoptosis rates. Moreover, strong G2/M arrest was detected suggesting important impaired proliferation after treatment. PLK1 mRNA expression level is not associated with prognosis in childhood ALL; however, considering the great variability observed in the sample and the in vitro experiments presented herein, BI 2536 treatment might serve as a promising therapeutic to enhance the efficacy of conventional treatment modalities in some childhood ALL cases.

A set of miRNAs that involve in the pathways of drug resistance and leukemic stem-cell differentiation is associated with the risk of relapse and glucocorticoid response in childhood ALL

Relapse is a major challenge in the successful treatment of childhood acute lymphoblastic leukemia (ALL). Despite intensive research efforts, the mechanisms of ALL relapse are still not fully understood. An understanding of the molecular mechanisms underlying treatment outcome, therapy response and the biology of relapse is required. In this study, we carried out a genome-wide microRNA (miRNA) microarray analysis to determine the miRNA expression profiles and relapse-associated miRNA patterns in a panel of matched diagnosis–relapse or diagnosis–complete remission (CR) childhood ALL samples. A set of miRNAs differentially expressed either in relapsed patients or at diagnosis compared with CR was further validated by quantitative real-time polymerase chain reaction in an independent sample set. Analysis of the predicted functions of target genes based on gene ontology ‘biological process’ categories revealed that the abnormally expressed miRNAs are associated with oncogenesis, classical multidrug resistance pathways and leukemic stem cell self-renewal and differentiation pathways. Several targets of the miRNAs associated with ALL relapse were experimentally validated, including FOXO3, BMI1 and E2F1. We further investigated the association of these dysregulated miRNAs with clinical outcome and confirmed significant associations for miR-708, miR-223 and miR-27a with individual relapse-free survival. Notably, miR-708 was also found to be associated with the in vivo glucocorticoid therapy response and with disease risk stratification. These miRNAs and their targets might be used to optimize anti-leukemic therapy, and serve as novel targets for development of new countermeasures of leukemia. This fundamental study may also contribute to establish the mechanisms of relapse in other cancers.

Adaptor molecules expression in normal lymphopoiesis and in childhood leukemia

Transmembrane adaptor proteins are key mediators of antigen receptor signaling in lymphocytes. By influencing proliferation and differentiation, these molecules might play a role in ethiopathogenesis of acute lymphoblastic leukemia (ALL). The aim of this study was to characterize expression of PAG, LAT, NTAL and LIME adaptors at the mRNA and protein levels in normal B- and T-precursors. Moreover, diagnostic samples of childhood ALL cases were analyzed. During normal lymphocyte development, some adaptors show significant dynamics (gradual decrease of NTAL and increase of LAT and LIME during the T-cell maturation, decrease of PAG in B-precursors, high levels of LIME in peripheral B-lymphocytes). Analysis of childhood ALL samples revealed that in B-cell precursor ALL, the TEL/AML1 subgroup have unique adaptor profile compared to other leukemias. Moreover, NTAL expression separates T lineage leukemias into two subgroups with good and poor response to initial prednisone therapy showing prognostic impact of this molecule in T-ALL.

Childhood Acute Lymphoblastic Leukemia: High Genomic Stability from Initial Diagnosis to Early Relapse.

Despite considerable improvements regarding treatment outcome about 20% of children with acute lymphoblastic leukemia (ALL) still suffer from recurrent disease. Especially patients with an early relapse occurring before 6 months of treatment cessation have a poor prognosis and, therefore, urgently need therapy optimization. As an initial step, detection of genetic aberrations that are associated with resistant disease and/or early relapse might be of great value as they could serve as prognostic markers to predict disease recurrence and, in addition, may increase our understanding of mechanisms underlying treatment resistance and early relapse. This knowledge may also help to identify new therapeutic strategies including targeted approaches. Surprisingly, almost no information is available on the genetic evolution from initial diagnosis to early relapse. As a first aim in the process of uncovering the pathomechanism of early relapse we wanted to find out if early relapse of childhood ALL reflects primarily resistant disease with little or no additional aberrations at relapse or represents evolution and selection of a new and more resistant clone possibly triggered through chemotherapy. Recently, high throughput technologies (single nucleotide polymorphism [SNP]–Arrays) have been developed to screen the whole genome with high resolution for submicroscopic, genetic alterations by simultaneously analyzing the SNP genotype and determining the copy number state. This prompted us to perform SNP-array analysis encompassing more than 100.000 SNPs in 20 childhood ALL samples collected at initial diagnosis and to compare them to their counterparts obtained at relapse. The analyzed patients belonged to the intermediate or high risk treatment group; 16 exhibited B-precursor and 4 T-precursor ALL. Exclusion criteria were translocations t(12;21), t(9;22) and (4;11) as well as pre-existing conditions (e.g. Down syndrome). At initial diagnosis, the leukemic sample of all patients already displayed a complex karyotype with multiple genetic lesions (average numbers per patient: 5.15 deletions, 1.35 amplifications, and 0.65 copy number neutral LOH (CNN-LOH)). Most alterations remained stable from initial diagnosis to relapse (99 of 103 (96%) initially observed deletions as well as 24 of 27 (89%) amplifications). Four deletions occurring in 3 different patients at initial diagnosis disappeared at relapse. Nevertheless, these patients had additional stable copy number alterations. Newly occurring aberrations at relapse were less frequent (average numbers per patient: 1.8 new deletions, 0.75 new amplifications, and 0.15 new CNN-LOH). In 3 patients genetic alterations at diagnosis and relapse were entirely identical. The most commonly affected chromosome was 9p (75% of samples). Fourteen patients had either heterozygous or homozygous deletions of CDKN2A at initial diagnosis and relapse with 2 patients progressing from heterozygous to homozygous deletions. PAX5 deletions (mainly heterozygous) were detectable in 9 patients at initial diagnosis and 10 patients at relapse. Other sides of recurrent deletions – mainly detected at relapse – included multiple areas of chromosome 7p (p21.3, p15.3, p14.3, p14.3-14.1, p14.1-p13) as well as chromosome 1q (deletion: q42.12-q44, amplification: q25.2-q31.1), chromosome 17p (deletion: p13.3-p11.2) and 17q (amplification: q21.2-q25.1). Potential pathogenetic implications of specific recurrent genetic lesions will be discussed. In conclusion, our results demonstrate a high degree of genomic stability from initial diagnosis to early relapse of childhood ALL. These results suggest that – at least in some patients – early relapse might already be predetermined by a resistant leukemic clone at the time of initial diagnosis.

Dexamethasone-induced apoptosis in acute lymphoblastic leukemia involves differential regulation of Bcl-2 family members

The mechanism of glucocorticoid -induced apoptosis is not fully understood and early predictive assays based on apoptotic markers for clinical outcome in acute lymphoblastic leukemia (ALL) are scarce. The aim of this study was to characterize the involvement of Bcl-2 family members and caspase activation in dexamethasone(Dex)-induced apoptosis in ALL. Primary childhood ALL samples, the pre-B ALL cell line RS(4;11), and the T-ALL cell line CCRF-CEM were used. The involvement of Bcl-2 family members was evaluated by flow cytometry, immunocytochemistry, and western and northern blotting. Apoptosis was analyzed by annexin V and TMRE staining. Caspase activity was evaluated by a fluorometric assay. Dex induced significant down-regulation of the anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, differential activation of the pro-apoptotic Bak and Bax, loss of Delta psi m and cytochrome c release. Dex-induced apoptosis also involved early activation of caspases 2 and -3. Inhibition of caspase activity did not, however, protect against Dex-induced Bak/Bax activation, loss of Delta psi m or cell death. In 12 primary ALL samples Dex-induced apoptosis was associated with activation of Bax (p=0.045) and down-regulation of Bcl-2 (p=0.016) and/or Bcl-xL (p=0.004). Furthermore, ex vivo Dex-sensitivity was associated with an early treatment response to polychemotherapy (p=0.026). The differential regulation of pro- and anti-apoptotic Bcl-2 family members appears to be a key event in the execution of Dex-induced apoptosis in ALL cell lines, and also indicates a role for these proteins in primary ALL cells.

Mutation in Genes Impacting on the RAS-RAF-MEK-ERK Pathway Are Found at High Incidence in Childhood Acute Lymphoblastic Leukemia at Both Diagnosis and at Relapse.

Deregulation of the RAS-RAF-MEK-ERK signalling cascade, often caused by activating mutations in the RAS family of G-proteins, is a recurrent event in haematological malignancies including childhood acute lymphoblastic leukemia (ALL). However, deregulation of this pathway can also arise from mutational events in genes encoding other proteins which impact on this pathway including FLT3, PTPN11 and BRAF and such mutations have recently been identified in childhood ALL. The combined incidence of mutations in these genes, along with the RAS family is not known, but may be significant.A comprehensive mutational screen of key exons of NRAS, KRAS2, FLT3, PTPN11 and BRAF was performed in 86 diagnostic and 49 relapse childhood ALL samples using denaturing high performance liquid chromatography (DHPLC) and direct sequencing and/or PCR product cloning. Using wild type cells spiked with cell lines bearing known mutations, the sensitivity of this screening assay was shown to be between 12–25%.At diagnosis the combined mutation incidence was 35% (30 from 86) made up of NRAS (n=16), KRAS2 (n=15), FLT3 (n=3), PTPN11 (n=2), with 4 patients having more than one mutation. At relapse, 25 % (12 from 49) of cases showed mutations and included NRAS (n=4), KRAS2 (n=6), FLT3 (n=1) and PTPN11 (n=1). No mutations were identified in BRAF. While at relapse the mutated blasts were always the predominant population, in 7 of the 30 diagnostic cases, only a minor subpopulation of blasts housed the mutation and included all of the FLT3 insertion/deletion mutants. In 2 relapsed cases with mutations in KRAS2, mutations were not detectable by DHPLC in the matched diagnostic samples but were apparent at low level when a more sensitive allele-specific PCR was employed (assay sensitivity between 0.1–1%).These data reveal a significant role of RAS-RAF-MEK-ERK pathway deregulation in leukemogenesis and implicate the pathway in some patients with progression to relapse. In addition, they highlight the genetic heterogeneity of leukemic blasts at diagnosis compared to those at relapse suggesting that clonal selection occurs in response to drug exposure. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for a significant proportion of children with relapsed ALL.

Thalidomide increases in vitro sensitivity of childhood acute lymphoblastic leukemia cells to prednisolone and cytarabine

Thalidomide is a derivative of glutamic acid with anti-angiogenic, anti-inflammatory, immunomodulatory and anti-cancer properties that was found to inhibit the production of tumor necrosis factor alpha in vitro, stimulate reactive oxygen species production, and inhibit vascular endothelial growth factor receptor in acute leukemias. The purpose of this study was to determine the in vitro activity of thalidomide as a single agent and in combination with prednisolone or cytarabine in childhood acute lymphoblastic leukemia (ALL). Bone marrow samples of 40 childhood ALL patients, normal lymphocytes of 9 healthy adults, and 3 lymphoid cell lines were evaluated for cytotoxicity of thalidomide (alone and in combination with prednisolone and cytarabine) using the MTT assay. Cell cycle analysis was performed by flow cytometry. Thalidomide as a single agent had weak antileukemic activity to the childhood ALL samples. However, in the presence of thalidomide the cytotoxicities of prednisolone and cytarabine were increased 3.3-fold (p<0.001) and 2.7-fold (p=0.002), respectively. Thalidomide increased apoptosis in lymphoblasts and modulated the cell-cycle arrest caused by prednisolone, but not that by cytarabine, in childhood ALL samples. Thalidomide increases in vitro the sensitivity of childhood ALL cells to prednisolone and cytarabine.

Ex vivo activity of thalidomide in childhood acute leukemia

Thalidomide is a drug with anti-angiogenic, anti-inflammatory, immunomodulatory and anti-cancer properties that were found to inhibit the production of TNF-α in vitro, stimulate reactive oxygen species production, and inhibit VEGFR in acute leukemias. Ex vivo activity of thalidomide as a single agent and in combination with prednisolone or cytarabine in childhood acute leukemias was analyzed. Forty samples of childhood acute lymphoblastic leukemia (ALL) and 13 acute myeloid leukemia (AML) were tested for cytotoxicity by the MTT assay and cell cycle phases by flow cytometry. Control studies were performed on 9 samples of normal lymphocytes and 4 cell lines. A weak anti-leukemic activity of thalidomide against childhood leukemic samples was observed. However, in the presence of thalidomide, cytotoxicity of prednisolone or cytarabine, increased 3.3-fold and 2.7-fold, respectively, in childhood ALL but was not changed in AML. Thalidomide increased apoptosis in lymphoblasts, and modulated cell cycle arrest caused by prednisolone but not cytarabine in childhood acute lymphoblastic leukemia samples. Thalidomide potentiated ex vivo sensitivity of childhood ALL cells to prednisolone and cytarabine, while no sensitization effect was observed in AML cells.

Single Monochrome Real-Time RT-PCR Assay for Identification, Quantification, and Breakpoint Cluster Region Determination of t(9;22) Transcripts

t(9;22) generates the BCR-ABL fusion gene, the hallmark of chronic myeloid leukemia (CML) but also found in acute lymphoblastic leukemia (ALL). Multiple chimeric transcripts translate to proteins of 190 or 210 kd and, rarely, 230 kd. CML typically carries p210 BCR-ABL while ALL is most often associated with p190. Detection and quantification of these fusion transcripts is useful in clinical management. We have exploited the unique melting profiles of these transcripts to design a new, simple, and cost-effective assay based on monochrome multiplex real-time RT-PCR for identification and quantification of each of these transcripts (b3-a2, b2-a2, and e1-a2) without further manipulation. The sensitivity of this assay was 10(-4) for e1-a2 and 10(-5) for b3-a2/b2-a2, which is appropriate for detection of minimal residual disease (MRD). Inter- and intra-assay variation was minimal. We applied this assay to assess the distribution of p190 and p210 in 260 childhood ALL samples from India. BCR-ABL was detected in 19 (7.3%), including one T-ALL. Eight patients (3.1%) demonstrated mBCR-ABL (p190) and 11 (4.2%) had MBCR-ABL (p210). Transcript levels varied markedly (up to 3000-fold) but e1-a2 were generally expressed at higher levels than b3/b2-a2 (P = 0.05). This simple real-time multiplex assay can thus be easily applied to monitor patients with ALL as well as CML.

Frameshift mutations in caspase-5 and other target genes in leukemia and lymphoma cell lines having microsatellite instability

Microsatellite instability (MSI) was examined in 49 lymphoid leukemia/lymphoma cell lines. Frameshift mutations within eight MSI target genes were analyzed in the 12 cell lines and nine childhood acute lymphoblastic leukemia (ALL) samples with MSI. Six cell lines (50%) showed mutations within the coding repeats of the BLM, caspase-5, TCF-4, and PTEN genes. No mutations were identified in the nine childhood ALL samples with MSI. This is the first report that describes mutations of these genes in hematological malignancies. Mutations, presumably caused by an abnormality of the DNA repair genes, may be selected for during the establishment of cell lines.

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells.

Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, K(i)s for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.

Raised blast glutathione levels are associated with an increased risk of relapse in childhood acute lymphocytic leukemia

AbstractA preliminary study has linked raised blast glutathione levels with chemoresistance in acute myeloid and lymphoblastic leukemia in adults and children. In this study, therefore, the relationship between leukemic blast glutathione levels and prognosis in childhood acute lymphoblastic leukemia (ALL) was investigated. A total of 77 childhood ALL samples were analyzed, 62 at initial presentation and 15 at relapse. A 20-fold interindividual variation in glutathione levels at presentation (median, 6.54 nmol/mg protein; range, 1.37 to 27.9) was demonstrated. The median level in T-lineage ALL was 2.3-fold higher than in B-lineage ALL (Mann-Whitney test,P < .0001). There was a significant correlation between presenting white cell count (WBC) and glutathione level (Spearman rank correlation coefficient, ρ = 0.45, P = .001). A high DNA index correlated with low glutathione levels (Mann-Whitney test,P = .013). There was no significant relationship between glutathione levels and in vitro drug sensitivity. Patients with glutathione levels above the median had a significantly greater risk of relapse (log-rank test statistic, 5.55; P = .018), and the overall survival rate was significantly reduced (log-rank test statistic, 4.38; P = .04). Multivariate analysis demonstrated that glutathione concentration was of independent prognostic value when assessed in conjunction with age, gender, WBC, and immunophenotype. The association of elevated blast glutathione levels with an increased risk of relapse suggests that glutathione-depleting agents may be of therapeutic value in patients who present with a high WBC.

Identification of three distinct regions of deletion on the long arm of chromosome 11 in childhood acute lymphoblastic leukemia.

Cytogenetic analysis of childhood acute lymphoblastic leukemia (ALL) identified deletions of chromosome arm 11q. These observations led us to analyse the loss of heterozygosity (LOH) of chromosome arm 11q in 113 primary childhood ALL samples using 14 microsatellite markers. LOH was found in 18 (16%) patients. Detailed examination identified three distinct regions of deletion. The first region is flanked by D11S901 and D11S1391 at 11q22-23 containing the ATM gene. Mutational analysis suggested that the altered gene in this region is not the ATM gene. The second region is flanked by D11S614 and D11S924 at 11q23 containing the MLL gene. The third region is flanked by D11S1356 and D11S614 at 11q23 containing the MLL gene. All the cases with LOH at MLL locus lacked detectable MLL gene rearrangements. In addition, 20 children have been studied both at initial diagnosis and relapse; none of the individuals who relapsed acquired LOH of 11q, suggesting that 11q deletions were infrequently involved in the progression of childhood ALL. Children with 11q LOH had a good response to induction chemotherapy (P=0.015). These data suggest that alterations of putative tumor suppressor genes on 11q are important events in development of childhood ALL. Our map provides important information toward cloning putative tumor suppressor genes associated with childhood ALL.

Comparison of the antileukemic activity in vitro of dexamethasone and prednisolone in childhood acute lymphoblastic leukemia

It is generally assumed that prednisolone (PRD) and dexamethasone (DXM) have equal glucocorticoid activity if PRD is given at sevenfold higher doses. Results of clinical studies of childhood acute lymphoblastic leukemia (ALL) suggested that DXM is more potent relative to PRD than assumed. The purpose of this study was to determine the relative antileukemic activity of PRD phosphate and DXM phosphate in 133 untreated childhood ALL samples in vitro, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. There was a marked variation in antileukemic activity of both agents among the patient samples. The median LC50 (drug concentration lethal to 50% of the ALL cells) for PRD phosphate was 3.50 μM, for DXM phosphate 0.20 μM. The individually calculated ratios of the LC50 values for PRD and DXM phosphate showed a large range from 0.7 to >500, with a median of 16.2. This 16-fold difference could not be explained by differences between these glucocorticoids in stability, hydrolysis into unesterified drug, adhesion to the wall of the microculture plates, or protein binding. ALL cells were cross-resistant to PRD and DXM phosphate (correlation coefficient = 0.85, P < 0.000001). We conclude that the in vitro antileukemic activity of DXM phosphate is median 16-fold higher than that of PRD phosphate, which contrasts to the generally assumed factor of 7. Based on the higher potency of DXM, and its more favorable pharmacokinetics as reported in the literature, DXM may be preferred to PRD as the glucocorticoid in the treatment of ALL. © 1996 Wiley-Liss, Inc.

Clinical and cell biological features related to cellular drug resistance of childhood acute lymphoblastic leukemia cells.

Several clinical and cell biological features, such as sex, age, leukemic cell burden, morphologic FAB type, and immunophenotype, have prognostic value in childhood acute lymphoblastic leukemia (ALL). The explanation for their prognostic significance is unclear, but might be related to cellular drug resistance. We prospectively studied the relation between the above mentioned features with resistance to 13 drugs in 144 childhood ALL samples obtained at initial diagnosis. The MTT assay was used for drug resistance testing. The interindividual differences in drug resistance were very large and exceeded those between the several subgroups. There was generally no significant relation between sex, leukemic cell burden, and FAB type with drug resistance. However, subgroups with a worse prognosis as defined by age (< 18 months and > 120 months at diagnosis) or immunophenotype (pro-B ALL and T-ALL) did show relatively resistant drug resistance profiles as compared to the subgroups with a better prognosis (age 18-120 months, common and pre-B ALL). Within the group of common and pre-B ALL and compared to the intermediate age-group, samples of the younger children were significantly more resistant to daunorubicin, mitoxantrone and teniposide, and samples of the older children were significantly more resistant to prednisolone and mercaptopurine. Pro-B ALL samples were significantly more resistant to 1-asparaginase and thioguanine, and T-ALL samples were significantly more resistant to prednisolone, dexamethasone, 1-asparaginase, vincristine, vindesine, daunorubicin, doxorubicin, teniposide, and ifosfamide, than the group of common and pre-B ALL cases. We conclude that the prognostic significance of age and immunophenotype in particular may be explained, at least partly, by its relation with resistance to certain drugs. The results of this study may be useful for future rational improvements of chemotherapeutic regimens in childhood ALL.